Process for making modulators of cystic fibrosis transmembrane conductance regulator

ABSTRACT

The invention provides a process for the preparation of a compound of Formula 1, 
     
       
         
         
             
             
         
       
     
     comprising coupling a carboxylic acid of Formula 2 
     
       
         
         
             
             
         
       
     
     with an aniline of Formula 3 
     
       
         
         
             
             
         
       
     
     in the presence of a coupling agent.

CLAIM OF PRIORITY

This application claims priority to three U.S. Provisional Applicationshaving Ser. Nos. 61/162,148, filed on Mar. 20, 2009; 61/246,303, filedon Sep. 28, 2009; and 61/248,565, filed on Oct. 5, 2009. Each of theforegoing Provisional Applications are hereby incorporated by referencein their entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a process for making modulators ofcystic fibrosis transmembrane conductance regulator (“CFTR”).

BACKGROUND OF THE INVENTION

Cystic fibrosis (CF) is a recessive genetic disease that affectsapproximately 30,000 children and adults in the United States andapproximately 30,000 children and adults in Europe. Despite progress inthe treatment of CF, there is no cure.

CF is caused by mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) gene that encodes an epithelial chlorideion channel responsible for aiding in the regulation of salt and waterabsorption and secretion in various tissues. Small molecule drugs, knownas potentiators that increase the probability of CFTR channel openingrepresent one potential therapeutic strategy to treat CF.

Specifically, CFTR is a cAMP/ATP-mediated anion channel that isexpressed in a variety of cells types, including absorptive andsecretory epithelia cells, where it regulates anion flux across themembrane, as well as the activity of other ion channels and proteins. Inepithelia cells, normal functioning of CFTR is critical for themaintenance of electrolyte transport throughout the body, includingrespiratory and digestive tissue. CFTR is composed of approximately 1480amino acids that encode a protein made up of a tandem repeat oftransmembrane domains, each containing six transmembrane helices and anucleotide binding domain. The two transmembrane domains are linked by alarge, polar, regulatory (R)-domain with multiple phosphorylation sitesthat regulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in cysticfibrosis (“CF”), the most common fatal genetic disease in humans. Cysticfibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with CF, mutations in CFTR endogenously expressed inrespiratory epithelia leads to reduced apical anion secretion causing animbalance in ion and fluid transport. The resulting decrease in aniontransport contributes to enhanced mucus accumulation in the lung and theaccompanying microbial infections that ultimately cause death in CFpatients. In addition to respiratory disease, CF patients typicallysuffer from gastrointestinal problems and pancreatic insufficiency that,if left untreated, results in death. In addition, the majority of maleswith cystic fibrosis are infertile and fertility is decreased amongfemales with cystic fibrosis. In contrast to the severe effects of twocopies of the CF associated gene, individuals with a single copy of theCF associated gene exhibit increased resistance to cholera and todehydration resulting from diarrhea—perhaps explaining the relativelyhigh frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, greater than 1000 diseasecausing mutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/app). The most prevalent mutationis a deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl ion channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases.

Accordingly, there is a need for modulators of CFTR activity, andcompositions thereof, which can be used to modulate the activity of theCFTR in the cell membrane of a mammal.

There is a need for methods of treating diseases caused by mutation inCFTR using such modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivocell membrane of a mammal.

There is also a need for processes for the preparation of compoundswhich modulate CFTR activity.

SUMMARY OF THE INVENTION

In general, the invention provides processes for the preparation ofcompounds useful as modulators of CFTR.

In one aspect, the invention provides a process for the preparation of acompound of Formula 1,

comprising coupling a carboxylic acid of Formula 2

with an aniline of Formula 3

in the presence of a coupling agent selected from the group consistingof 2-chloro-1,3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU,2-chloro-4,6-dimethoxy-1,3,5-triazine, HATU, HOBT/EDC, and T3P®.

Each R₂ and R₄ is independently selected from hydrogen, CN, CF₃, halo,C₁₋₆ straight or branched alkyl, 3-12 membered cycloaliphatic, phenyl,C₅₋₁₀ heteroaryl or C₃₋₇ heterocyclic, wherein said heteroaryl orheterocyclic has up to 3 heteroatoms selected from O, S, or N, and eachC₁₋₆ straight or branched alkyl, 3-12 membered cycloaliphatic, phenyl,C₅₋₁₀ heteroaryl or C₃₋₇ heterocyclic is independently and optionallysubstituted with up to three substituents selected from —OR, —CF₃,—OCF₃, SR′, S(O)R′, SO₂R′, —SCF₃, halo, CN, —COOR′, —COR—,—O(CH₂)₂N(R′)(R′), —O(CH₂)N(R′)(R′), —CON(R′)(R′), —(CH₂)₂OR′,—(CH₂)OR′, CH₂CN, optionally substituted phenyl or phenoxy, —N(R′)(R′),—NR′C(O)OR′, —NR′C(O)R′, —(CH₂)₂N(R′)(R′), or —(CH₂)N(R′)(R′).

Each R₅ is independently selected from hydrogen, —OH, NH₂, CN, CHF₂,NHR′, N(R′)₂, —NHC(O)R′, NHC(O)OR′, NHSO₂R′, —OR′, OC(O)OR′, OC(O)NHR′,OC(O)NR′₂, CH₂OH, CH₂N(R′)₂, C(O)OR′, SO₂NHR′, SO₂N(R′)₂, orCH₂NHC(O)OR′.

Or R₄ and R₅ are taken together form a 5-7 membered ring containing 0-3three heteroatoms selected from N, O, or S, wherein said ring isoptionally substituted with up to three R₃ substituents.

Each X is independently a bond or is an optionally substituted C₁₋₆alkylidene chain wherein up to two methylene units of X are optionallyand independently replaced by —CO—, —CS—, —COCO—, —CONR′—, —CONR′NR′—,—CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—, —NR′NR′, —NR′NR′CO—,—NR′CO—, —S—, —SO, —SO₂—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—.

Each R^(x) is independently R′, halo, NO₂, CN, CF₃, or OCF₃.

y is an integer from 0-4.

Each R′ is independently selected from hydrogen or an optionallysubstituted group selected from a C₁₋₈ aliphatic group, a 3-8-memberedsaturated, partially unsaturated, or fully unsaturated monocyclic ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or an 8-12 membered saturated, partially unsaturated, or fullyunsaturated bicyclic ring system having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; or two occurrences of R′ aretaken together with the atom(s) to which they are bound to form anoptionally substituted 3-12 membered saturated, partially unsaturated,or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatomsindependently selected from N, O, or S.

Each R₃ is independently —C₁-C₃ alkyl, C₁-C₃ perhaloalkyl, —O(C₁-C₃alkyl), —CF₃, —OCF₃, —SCF₃, —F, —Cl, —Br, —COOR′, —COR′,—O(CH₂)₂N(R′)(R′), —O(CH₂)N(R′)(R′), —CON(R′)(R′), —(CH₂)₂OR′,—(CH₂)OR′, optionally substituted monocyclic or bicyclic aromatic ring,optionally substituted arylsulfone, optionally substituted 5-memberedheteroaryl ring, —N(R′)(R′), —(CH₂)₂N(R′)(R′), or —(CH₂)N(R′)(R′).

Embodiments of this aspect include one or more of the followingfeatures. R₅ is independently —OC(O)OR′, —OC(O)NHR′, or —OC(O)N(R′)₂,and R′ is not hydrogen; at least one of R₄ or R₂ is independently a C₁₋₆straight or branched alkyl which is substituted with —COOR′ or—CON(R′)(R′), and R′ is not hydrogen. The process further comprisescleaving the —OC(O)OR′, —OC(O)NHR′, or —OC(O)N(R′)₂ group to form —OH.The process further comprises hydrolyzing each —COOR′, or —CON(R′)₂group to form —COOH. The hydrolysis is performed by treating a compoundof Formula 1 with an alcoholic solvent in the presence of base such asNaOH, KOH or sodium methoxide. The alcoholic solvent used in thehydrolysis is methanol. The coupling a compound of Formula 2 and acompound of Formula 3 to produce a compound of Formula 1 is performed inthe presence of a base such as K₂CO₃, Et₃N, NMM, pyridine or DIEA. Thecoupling a compound of Formula 2 and a compound of Formula 3 to producea compound of Formula 1 is performed in the presence of a solvent suchas EtOAc, IPAc, THF, MEK, NMP, acetonitrile, DMF, or2-methyltetrahydrofuran. The coupling a compound of Formula 2 and acompound of Formula 3 to produce a compound of Formula 1 is performed ata reaction temperature which is maintained between about 10° C. and 78°C. such as between about 20° C. and 30° C., between about 40° C. and 50°C., and between about 42° C. and 53° C. The coupling reaction is stirredfor at least 2 hours such as for at least 70 hours or for at least 3days.

In some embodiments, R₅ is independently —OC(O)OR′, —OC(O)NHR′, or—OC(O)N(R′)₂, and R′ is not hydrogen; and each of R₂ and R₄ isindependently selected from hydrogen, CF₃, C₁-C₆ straight or branchedalkyl, 3-12 membered cycloaliphatic or phenyl.

In some further embodiments, R₅ is independently —OC(O)OR′, and R′ isnot hydrogen; and each of R₂ and R₄ is independently C₁-C₆ straight orbranched alkyl or 3-12 membered cycloaliphatic.

In some embodiments, R₂ and R₄ are t-butyl.

In another aspect, the invention provides a process for the preparationof compound 27

comprising:

(a) coupling compound 26

with compound 13

in the presence of EDCI, HOBT and DIEA using DMF as the solvent, whereinthe reaction temperature is maintained between about 20° C. and 30° C.,and the reaction is allowed proceed for at least 70 hours, to producecompound 14

and

(b) treating compound 14 with KOH in methanol.

In still another aspect, the invention provides a process for thepreparation of compound 28

comprising:

(a) coupling compound 26

with compound 20

in the presence of HATU and DIEA using acetonitrile as the solvent,wherein the reaction temperature is maintained between about 40° C. and50° C., and wherein the reaction is allowed proceed for at least 3 days,to produce compound 21

and

(b) treating compound 21 with NaOH in methanol.

In yet another aspect, the invention provides a process for thepreparation of compound 34

comprising:

(a) coupling compound 26

with compound 32

in the presence of T3P® and pyridine using 2-methyl tetrahydrofuran asthe solvent, wherein the reaction temperature is maintained betweenabout 42° C. and 53 OC, and wherein the reaction is allowed proceed forat least 2 hours, to produce compound 33

(b) treating compound 33 with NaOMe/MeOH in 2-methyl tetrahydrofuran.

In one embodiment, the method further includes the step of forming aslurry of compound 34 in a mixture of acetonitrile and water, whereinthe solid form of compound 34 is converted to Compound 34.

Embodiments of the forgoing aspect include one or more of the followingfeatures. The process further comprises dissolving Compound 34 in abiphasic solution of 2-methyltetrahydrofuran and 0.1N HCl, which isstirred. The process further comprises separating the organic phase fromthe biphasic solution. The process further comprises filtering andremoving solid matter from the organic phase. The process furthercomprises reducing the volume of the organic phase by approximately 50%using distillation. The process further comprises performing thrice theprocedure of: adding MeOAc, EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF),Et₂O or methyl-t-butyl ether (MTBE) to the organic phase until thevolume of the organic phase increases by 100% and reducing the volume ofthe organic phase by 50% using distillation. The process furthercomprises adding MeOAc, EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF),Et₂O or methyl-t-butyl ether (MTBE) to the organic phase until thevolume of the organic phase increases by 100%. The process furthercomprises heating the organic phase to reflux temperature, andmaintaining said reflux temperature for a time at least about 5 hours.The process further comprises cooling the organic phase to a temperaturebetween −5° C. and 5° C. over a time period of 4.5 hours to 5.5 hours.

In still another aspect, the invention provides compounds produced byany process described herein.

In a further aspect, the invention provides a pharmaceutical compositioncomprising a compound produced by any process described herein.

In still a further aspect, the invention provides a method of modulatingCFTR activity in a biological sample comprising the step of contactingsaid biological sample with a compound produced by any process describedherein.

In another aspect, the invention also provides a method of treating orlessening the severity of a disease in a patient comprisingadministering to said patient one of the compositions as defined herein,and said disease is selected from cystic fibrosis, asthma, smoke inducedCOPD, chronic bronchitis, rhinosinusitis, constipation, pancreatitis,pancreatic insufficiency, male infertility caused by congenitalbilateral absence of the vas deferens (CBAVD), mild pulmonary disease,idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA),liver disease, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington's, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease, Osteoporosis, Osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, and PrimaryCiliary Dyskinesia (PCD), a term for inherited disorders of thestructure and/or function of cilia, including PCD with situs inversus(also known as Kartagener syndrome), PCD without situs inversus andciliary aplasia.

In certain embodiments, the disease is cystic fibrosis.

In another aspect, the invention provides a kit for use in measuring theactivity of CFTR or a fragment thereof in a biological sample in vitroor in vivo, comprising:

-   -   i. a composition comprising a compound produced by any process        described herein; and    -   ii. instructions for:        -   a. contacting the composition with the biological sample;            and        -   b. measuring the activity of said CFTR or a fragment            thereof.

In certain embodiments, the kit further comprises instructions for:

-   -   i. contacting an additional compound with the biological sample;    -   ii. measuring the activity of said CFTR or a fragment thereof in        the presence of said additional compound; and    -   iii. comparing the activity of the CFTR in the presence of the        additional compound with the density of the CFTR in the presence        of a composition of Formula 1.

Advantageously, the invention provides processes for the synthesis ofcompounds useful as modulators of CFTR in higher yield and in higherpurity relative to known processes.

DETAILED DESCRIPTION I. Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “ABC-transporter” as used herein means an ABC-transporterprotein or a fragment thereof comprising at least one binding domain,wherein said protein or fragment thereof is present in vivo or in vitro.The term “binding domain” as used herein means a domain on theABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. etal., J. Gen. Physiol. (1998): 111(3), 477-90.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see,e.g., http://www.genet.sickkids.on.calcftr/, for CFTR mutations).

The term “modulating” as used herein means increasing or decreasing by ameasurable amount.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75th Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausalito: 1999, and “March'sAdvanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J.,John Wiley & Sons, New York: 2001, the entire contents of which arehereby incorporated by reference.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent.

Unless otherwise indicated, an optionally substituted group may have asubstituent at each substitutable position of the group, and when morethan one position in any given structure may be substituted with morethan one substituent selected from a specified group, the substituentmay be either the same or different at every position. Combinations ofsubstituents envisioned by this invention are preferably those thatresult in the formation of stable or chemically feasible compounds.

The term “stable”, as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and preferably their recovery, purification, anduse for one or more of the purposes disclosed herein. In someembodiments, a stable compound or chemically feasible compound is onethat is not substantially altered when kept at a temperature of 40° C.or less, in the absence of moisture or other chemically reactiveconditions, for at least a week.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle”, “cycloaliphatic”, or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-20 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-10aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-8 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-6 aliphatic carbon atoms, and in yet other embodimentsaliphatic groups contain 1-4 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃₋₈ hydrocarbon or bicyclic or tricyclic C₈₋₁₄hydrocarbon that is completely saturated or that contains one or moreunits of unsaturation, but which is not aromatic, that has a singlepoint of attachment to the rest of the molecule wherein any individualring in said bicyclic ring system has 3-7 members. Suitable aliphaticgroups include, but are not limited to, linear or branched, substitutedor unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof suchas (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.Suitable cycloaliphatic groups include cycloalkyl, bicyclic cycloalkyl(e.g., decalin), bridged bicycloalkyl such as norbornyl or[2.2.2]bicyclo-octyl, or bridged tricyclic such as adamantyl.

The term “heteroaliphatic”, as used herein, means aliphatic groupswherein one or two carbon atoms are independently replaced by one ormore of oxygen, sulfur, nitrogen, phosphorus, or silicon.Heteroaliphatic groups may be substituted or unsubstituted, branched orunbranched, cyclic or acyclic, and include “heterocycle”,“heterocyclyl”, “heterocycloaliphatic”, or “heterocyclic” groups.

The term “heterocycle”, “heterocyclyl”, “heterocycloaliphatic”, or“heterocyclic” as used herein means non-aromatic, monocyclic, bicyclic,or tricyclic ring systems in which one or more ring members is anindependently selected heteroatom. In some embodiments, the“heterocycle”, “heterocyclyl”, “heterocycloaliphatic”, or “heterocyclic”group has three to fourteen ring members in which one or more ringmembers is a heteroatom independently selected from oxygen, sulfur,nitrogen, or phosphorus, and each ring in the system contains 3 to 7ring members.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

The term “alkoxy”, or “thioalkyl”, as used herein, refers to an alkylgroup, as previously defined, attached to the principal carbon chainthrough an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.

The terms “haloaliphatic” and “haloalkoxy” means aliphatic or alkoxy, asthe case may be, substituted with one or more halo atoms. The term“halogen” or “halo” means F, Cl, Br, or I. Examples of haloaliphaticinclude —CHF₂, —CH₂F, —CF₃, —CF₂—, or perhaloalkyl, such as, —CF₂CF₃.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains 3 to 7 ring members. The term“aryl” may be used interchangeably with the term “aryl ring”. The term“aryl” also refers to heteroaryl ring systems as defined herein below.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms, and whereineach ring in the system contains 3 to 7 ring members. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic”.

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) orheteroaryl (including heteroaralkyl and heteroarylalkoxy and the like)group may contain one or more substituents. Suitable substituents on theunsaturated carbon atom of an aryl or heteroaryl group are selected fromhalo; —R^(o); —OR^(o); —SR^(o); 1,2-methylene-dioxy; 1,2-ethylenedioxy;phenyl (Ph) optionally substituted with R^(o); —O(Ph) optionallysubstituted with R^(o); —(CH₂)₁₋₂(Ph), optionally substituted withR^(o); —CH═CH(Ph), optionally substituted with R^(o); —NO₂; —CN;—N(R^(o))₂; —NR^(o)C(O)R^(o); —NR^(o)C(O)N(R^(o))₂; —NR^(o)CO₂R^(o);—NR^(o)NR^(o)C(O)R^(o); —NR^(o)NR^(o)C(O)N(R^(o))₂;—NR^(o)NR^(o)CO₂R^(o); —C(O)C(O)R; —C(O)CH₂C(O)R^(o); —CO₂R^(o); —C(O)R;—C(O)N(R^(o))₂; —OC(O)N(R^(o))₂; —S(O)₂R^(o); —SO₂N(R^(o))₂; —S(O)R^(o);—NR^(o)SO₂N(R^(o))₂; —NR^(o)SO₂R^(o); —C(═S)N(R^(o))₂;—C(═NH)—N(R^(o))₂; or —(CH₂)₀₋₂NHC(O)R^(o) wherein each independentoccurrence of R^(o) is selected from hydrogen, optionally substitutedC₁₋₆ aliphatic, an unsubstituted 5-6 membered heteroaryl or heterocyclicring, phenyl, —O(Ph), or —CH₂(Ph), or, notwithstanding the definitionabove, two independent occurrences of R^(o), on the same substituent ordifferent substituents, taken together with the atom(s) to which eachR^(o) group is bound, form a 3-8-membered cycloalkyl, heterocyclyl,aryl, or heteroaryl ring having 0-3 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Optional substituents on the aliphaticgroup of R^(o) are selected from NH₂, NH(C₁₋₄aliphatic),N(C₁₋₄aliphatic)₂, halo, C₁₋₄ aliphatic, OH, O(C₁₋₄ aliphatic), NO₂, CN,CO₂H, CO₂(C₁₋₄ aliphatic), O(halo C₁₋₄ aliphatic), or haloC₁₋₄aliphatic, wherein each of the foregoing C₁₋₄ aliphatic groups of R^(o)is unsubstituted.

An aliphatic or heteroaliphatic group, or a non-aromatic heterocyclicring may contain one or more substituents. Suitable substituents on thesaturated carbon of an aliphatic or heteroaliphatic group, or of anon-aromatic heterocyclic ring are selected from those listed above forthe unsaturated carbon of an aryl or heteroaryl group and additionallyinclude the following: ═O, ═S, ═NNHR*, ═NN(R*)₂, ═NNHC(O)R*,═NNHCO₂(alkyl), ═NNHSO₂(alkyl), or ═NR*, where each R* is independentlyselected from hydrogen or an optionally substituted C₁₋₆ aliphatic.Optional substituents on the aliphatic group of R* are selected fromNH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halo, C₁₋₄ aliphatic, OH,O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄ aliphatic), O(halo C₁₋₄aliphatic), or halo(C₁₋₄ aliphatic), wherein each of the foregoing C₁₋₄aliphatic groups of R* is unsubstituted.

Optional substituents on the nitrogen of a non-aromatic heterocyclicring are selected from —R⁺, —N(R⁺)₂, —C(O)R⁺, —CO₂R⁺, —C(O)C(O)R⁺,—C(O)CH₂C(O)R⁺, —SO₂R⁺, —SO₂N(R)₂, —C(═S)N(R⁺)₂, —C(═NH)—N(R⁺)₂, or—NR⁺SO₂R⁺; wherein R⁺ is hydrogen, an optionally substituted C₁₋₆aliphatic, optionally substituted phenyl, optionally substituted —O(Ph),optionally substituted —CH₂(Ph), optionally substituted —(CH₂)₁₋₂(Ph);optionally substituted —CH═CH(Ph); or an unsubstituted 5-6 memberedheteroaryl or heterocyclic ring having one to four heteroatomsindependently selected from oxygen, nitrogen, or sulfur, or,notwithstanding the definition above, two independent occurrences of R⁺,on the same substituent or different substituents, taken together withthe atom(s) to which each R⁺ group is bound, form a 3-8-memberedcycloalkyl, heterocyclyl, aryl, or heteroaryl ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur.Optional substituents on the aliphatic group or the phenyl ring of R⁺are selected from NH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halo,C₁₋₄ aliphatic, OH, O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄aliphatic), O(halo C₁₋₄ aliphatic), or halo(C₁₋₄ aliphatic), whereineach of the foregoing C₁₋₄ aliphatic groups of R⁺ is unsubstituted.

The term “alkylidene chain” refers to a straight or branched carbonchain that may be fully saturated or have one or more units ofunsaturation and has two points of attachment to the rest of themolecule. The term “spirocycloalkylidene” refers to a carbocyclic ringthat may be fully saturated or have one or more units of unsaturationand has two points of attachment from the same ring carbon atom to therest of the molecule.

The term “slurry,” as used herein, is defined as a mixture comprising asolid and a liquid, wherein the solid is, at most, partially soluble inthe liquid. The term “slurrying” or “slurried,” as used herein (example,“the solid product was slurried for 24 hours”), is defined as the act ofcreating a slurry, and stirring said slurry for a length of time.

The term “protecting group” (PG) as used herein, represents those groupsintended to protect a functional group, such as, for example, analcohol, amine, carboxyl, carbonyl, etc., against undesirable reactionsduring synthetic procedures. Commonly used protecting groups aredisclosed in Greene and Wuts, Protective Groups in Organic Synthesis,3rd Edition (John Wiley & Sons, New York, 1999), which is incorporatedherein by reference. Examples of nitrogen protecting groups includeacyl, aroyl, or carbamyl groups such as formyl, acetyl, propionyl,pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, ca-chlorobutyryl,benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and chiralauxiliaries such as protected or unprotected D, L or D, L-amino acidssuch as alanine, leucine, phenylalanine and the like; sulfonyl groupssuch as benzenesulfonyl, p-toluenesulfonyl and the like; carbamategroups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike, arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyland the like and silyl groups such as trimethylsilyl and the like.Another exemplary N-protecting group is tert-butyloxycarbonyl (Boc).

Examples of useful protecting groups for acids are substituted alkylesters such as 9-fluorenylmethyl, methoxymethyl, methylthiomethyl,tetrahydropyranyl, tetrahydrofuranyl, methoxyethoxymethyl,2-(trimethylsilyl)ethoxymethyl, benzyloxymethyl, pivaloyloxymethyl,phenylacetoxymethyl, triisopropropylsysilylmethyl, cyanomethyl, acetol,phenacyl, substituted phenacyl esters, 2,2,2-trichloroethyl,2-haloethyl, co-chloroalkyl, 2-(trimethylsilyl)ethyl, 2-methylthioethyl,t-butyl, 3-methyl-3-pentyl, dicyclopropylmethyl, cyclopentyl,cyclohexyl, allyl, methallyl, cynnamyl, phenyl, silyl esters, benzyl andsubstituted benzyl esters, 2,6-dialkylphenyl esters such aspentafluorophenyl, 2,6-dialkylpyhenyl. Other protecting groups for acidsare methyl or ethyl esters.

Methods of adding (a process generally referred to as “protection”) andremoving (process generally referred to as “deprotection”) such amineand acid protecting groups are well-known in the art and available, forexample in P. J. Kocienski, Protecting Groups, Thieme, 1994, which ishereby incorporated by reference in its entirety and in Greene and Wuts,Protective Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons,New York, 1999).

Examples of suitable solvents that may be used in this invention are,but not limited to water, methanol, dichloromethane (DCM), acetonitrile,dimethylformamide (DMF), methyl acetate (MeOAc), ethyl acetate (EtOAc),isopropyl acetate (IPAc), t-butyl acetate (t-BuOAc), isopropyl alcohol(IPA), tetrahydrofuran (THF), methyl ethyl ketone (MEK), t-butanol,diethyl ether (Et₂O), methyl-t-butyl ether (MTBE), 1,4-dioxane andN-methyl pyrrolidone (NMP).

Examples of suitable coupling agents that may be used in this inventionare, but not limited to 1-(3-(dimethylamino)propyl)-3-ethyl-carbodiimidehydrochloride (EDCI),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), 1-hydroxybenzotriazole (HOBT),2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU), 2-chloro-1,3-dimethyl-2-imidazoliumtetrafluoroborate,1-H-benzotriazolium-1-[bis(dimethylamino)methylene]-5-chlorohexafluorophosphate(HCTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine, and 2-propane phosphonicanhydride (T3P®).

Examples of suitable bases that may be used in this invention are, butnot limited to potassium carbonate (K₂CO₃), N-methylmorpholine (NMM),triethylamine (Et₃N; TEA), diisopropyl-ethyl amine (i-Pr₂EtN; DIEA),pyridine, potassium hydroxide (KOH), sodium hydroxide (NaOH), and sodiummethoxide (NaOMe; NaOCH₃).

In some embodiments, two independent occurrences of R^(o), as depictedin the structure below, are taken together with the atom(s) to whichthey are attached to form a 3-8-membered cycloalkyl, heterocyclyl, aryl,or heteroaryl ring having 0-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. Exemplary rings that are formed when twoindependent occurrences of R^(o) are taken together with the atom(s) towhich they are attached include, but are not limited to the following:a) two independent occurrences of R^(o) that are bound to the same atomand are taken together with that atom to form a ring, for example,N(R^(o))₂, where both occurrences of R^(o) are taken together with thenitrogen atom to form a piperidin-1-yl, piperazin-1-yl, ormorpholin-4-yl group; and b) two independent occurrences of R^(o) thatare bound to different atoms and are taken together with both of thoseatoms to form a ring, for example where a phenyl group is substitutedwith two occurrences of OR^(o)

these two occurrences of R^(o) are taken together with the oxygen atomsto which they are bound to form a fused 6-membered oxygen containingring:

It will be appreciated that a variety of other rings can be formed whentwo independent occurrences of R^(o) are taken together with the atom(s)to which each variable is bound and that the examples detailed above arenot intended to be limiting.

Ring substituents on, for example, mono and poly aryl, aliphatic,heteroaliphatic ring systems can be attached on any ring position forwhich it is chemically feasible to attach a substituent.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention. That is when R^(x)—X— in a compound of Formula 1 ishydrogen, said compound of Formula 1 may exist as a tautomer:

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C or ¹⁴C are withinthe scope of this invention. Such compounds are useful, for example, asanalytical tools, probes in biological assays or as therapeutic agents.

II. Processes of the Invention

In general, the invention provides processes for the synthesis ofcompounds useful as modulators of CFTR.

In some embodiments, the invention provides a process for thepreparation of a compound having the structure

In some embodiments, the invention provides a process for thepreparation of a compound having the structure

In some embodiments, the invention provides a process for thepreparation of a compound having the structure

In one aspect, the invention provides a process for the preparation of acompound of Formula 1,

comprising coupling a carboxylic acid of Formula 2

with an aniline of Formula 3

in the presence of a coupling agent selected from the group consistingof 2-chloro-1,3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU,2-chloro-4,6-dimethoxy-1,3,5-triazine, HATU, HOBT/EDC, and T3P®.

Each R₂ and R₄ is independently selected from hydrogen, CN, CF₃, halo,C₁₋₆ straight or branched alkyl, 3-12 membered cycloaliphatic, phenyl,C₅₋₁₀ heteroaryl or C₃₋₇ heterocyclic, wherein said heteroaryl orheterocyclic has up to 3 heteroatoms selected from O, S, or N, and eachC₁₋₆ straight or branched alkyl, 3-12 membered cycloaliphatic, phenyl,C₅₋₁₀ heteroaryl or C₃₋₇ heterocyclic is independently and optionallysubstituted with up to three substituents selected from —OR, —CF₃,—OCF₃, SR′, S(O)R′, SO₂R′, —SCF₃, halo, CN, —COOR′, —COR—,—O(CH₂)₂N(R′)(R′), —O(CH₂)N(R′)(R′), —CON(R′)(R′), —(CH₂)₂OR′,—(CH₂)OR′, CH₂CN, optionally substituted phenyl or phenoxy, —N(R′)(R′),—NR′C(O)OR′, —NR′C(O)R′, —(CH₂)₂N(R′)(R′), or —(CH₂)N(R′)(R′).

Each R₅ is independently selected from hydrogen, —OH, NH₂, CN, CHF₂,NHR′, N(R′)₂, —NHC(O)R′, NHC(O)OR′, NHSO₂R′, —OR′, OC(O)OR′, OC(O)NHR′,OC(O)NR′₂, CH₂OH, CH₂N(R′)₂, C(O)OR′, SO₂NHR′, SO₂N(R′)₂, orCH₂NHC(O)OR′.

Or, R₄ and R₅ are taken together form a 5-7 membered ring containing 0-3three heteroatoms selected from N, O, or S, wherein said ring isoptionally substituted with up to three R₃ substituents.

Each X is independently a bond or is an optionally substituted C₁₋₆alkylidene chain wherein up to two methylene units of X are optionallyand independently replaced by —CO—, —CS—, —COCO—, —CONR′—, —CONR′NR′—,—CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—, —NR′NR′, —NR′NR′CO—,—NR′CO—, —S—, —SO, —SO₂—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—.

Each R^(X) is independently R′, halo, NO₂, CN, CF₃, or OCF₃. y is aninteger from 0-4. Each R′ is independently selected from hydrogen or anoptionally substituted group selected from a C₁₋₈ aliphatic group, a3-8-membered saturated, partially unsaturated, or fully unsaturatedmonocyclic ring having 0-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or an 8-12 membered saturated, partiallyunsaturated, or fully unsaturated bicyclic ring system having 0-5heteroatoms independently selected from nitrogen, oxygen, or sulfur; ortwo occurrences of R′ are taken together with the atom(s) to which theyare bound to form an optionally substituted 3-12 membered saturated,partially unsaturated, or fully unsaturated monocyclic or bicyclic ringhaving 0-4 heteroatoms independently selected from N, O, or S.

Each R₃ is independently —C₁₋₃ alkyl, C₁₋₃ perhaloalkyl, —O(C₁₋₃ alkyl),—CF₃, —OCF₃, —SCF₃, —F, —Cl, —Br, or —COOR′, —COR′, —O(CH₂)₂N(R′)(R′),—O(CH₂)N(R′)(R′), —CON(R′)(R′), —(CH₂)₂OR′, —(CH₂)OR′, optionallysubstituted monocyclic or bicyclic aromatic ring, optionally substitutedarylsulfone, optionally substituted 5-membered heteroaryl ring,—N(R′)(R′), —(CH₂)₂N(R′)(R′), or —(CH₂)N(R′)(R′).

In one embodiment, R₅ is independently —OC(O)OR′, —OC(O)NHR′, or—OC(O)N(R′)₂, and R′ is not hydrogen. In certain instances R₅ is—OC(O)OR′ and R′ is not hydrogen. In other instances, R₅ is —OC(O)NHR′and R′ is not hydrogen. In still other instances, R₅ is —OC(O)N(R′)₂ andR′ is not hydrogen.

In one embodiment, the process further comprises cleaving the —OC(O)OR′,—OC(O)NHR′, or —OC(O)N(R′)₂R₅ group to form —OH. The cleavage isperformed by treating a compound of Formula 1 containing the —OC(O)OR′,—OC(O)NHR′, or —OC(O)N(R′)₂R₅ group with an alcoholic solvent in thepresence of base such as NaOH, KOH or sodium methoxide. The alcoholicsolvent used in the cleavage reaction is methanol, ethanol, isopropylalcohol or t-butanol.

In another embodiment, at least one of R₄ or R₂ is independently a C₁-C₆straight or branched alkyl which is substituted with —COOR′ or—CON(R′)₂, and R′ is not hydrogen. In certain instances, one of R₄ or R₂is —COOR′ and R′ is not hydrogen. In other instances, one of R₄ or R₂ is—CON(R′)₂ and R′ is not hydrogen.

In one embodiment, the process further comprises hydrolyzing the —COOR′or —CON(R′)₂ on at least one of R₄ and R₂. The hydrolysis is performedby treating a compound of Formula 1 containing the —COOR′ or —CON(R′)₂group on at least one of R₄ and R₂ with an alcoholic solvent in thepresence of base such as NaOH, KOH or sodium methoxide. The alcoholicsolvent used in the hydrolysis is methanol, ethanol, isopropyl alcoholor t-butanol.

In another embodiment, at least one of R₄ or R₂ is independently a C₁₋₆straight or branched alkyl which is substituted with —COOR′ or —CON(R′)₂and R₅ is independently —OC(O)OR′, —OC(O)NHR′, or —OC(O)N(R′)₂, and eachR′ is not hydrogen.

In one embodiment, the process further comprises hydrolyzing the —COOR′or —CON(R′)₂ on at least one of R₄ and R₂ and cleaving the —OC(O)OR′,—OC(O)NHR′, or —OC(O)N(R′)₂R₅ group. The hydrolysis/cleavage reaction isperformed by treating a compound of Formula 1 containing the —COOR′ or—CON(R′)₂ group on at least one of R₄ and R₂ and —OC(O)OR′, —OC(O)NHR′,or —OC(O)N(R′)₂R₅ group with an alcoholic solvent in the presence ofbase such as NaOH, KOH or sodium methoxide. The alcoholic solvent usedin the hydrolysis/cleavage reaction is methanol, ethanol, isopropylalcohol or t-butanol.

In another embodiment, the coupling of the carboxylic acid of Formula 2and the aniline of Formula 3 is performed in the presence of a base suchas K₂CO₃, Et₃N, N-methylmorpholine (NMM), pyridine or DIEA.

In another embodiment, the coupling of the carboxylic acid of Formula 2and the aniline of Formula 3 is performed in the presence of pyridine orDIEA.

In yet another embodiment, the coupling of the carboxylic acid ofFormula 2 and the aniline of Formula 3 is performed in the presence of asolvent such as EtOAc, IPAc, THF, MEK, NMP, acetonitrile, DMF, or2-methyltetrahydrofuran.

In further embodiments, the coupling of the carboxylic acid of Formula 2and the aniline of Formula 3 is performed at a reaction temperaturewhich is maintained between 10° C. and 78° C. such as between about 20°C. and 30° C., between about 40° C. and 50° C., and between about 42° C.and 53 OC.

In still further embodiments, the coupling reaction is stirred for atleast 2 hours such as for at least 8 hours, for at least 70 hours or forat least 3 days.

In another embodiment, y is 0.

In still other embodiments, R₂ is tert-butyl.

In some embodiments, R₅ is independently —OC(O)OR′, —OC(O)NHR′, or—OC(O)N(R′)₂, and R′ is not hydrogen; and each of R₂ and R₄ isindependently selected from hydrogen, CF₃, C₁-C₆ straight or branchedalkyl, 3-12 membered cycloaliphatic or phenyl.

In some embodiments, R₅ is independently —OC(O)OR′, —OC(O)NHR′, or—OC(O)N(R′)₂, and R′ is not hydrogen; and each of R₂ and R₄ isindependently selected from C₁-C₆ straight or branched alkyl.

In some embodiments, R₅ is independently —OC(O)OR′, —OC(O)NHR′, or—OC(O)N(R′)₂, and R′ is not hydrogen; and each of R₂ and R₄ isindependently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, t-butyl, n-pentyl, or n-hexyl.

In some embodiments, R₂ and R₄ are t-butyl.

In one embodiment, the invention provides a process for the preparationof a compound of Formula 5

by reacting a compound of Formula 6

with a reagent capable of causing a protecting group to be attached tothe phenolic oxygen of a compound of Formula 6 in the presence of asolvent, thereby producing a compound of Formula 7

which is nitrated to form a compound of Formula 8

which is then reduced to give a compound of Formula 5, wherein PG is aprotecting group and R₄ and R₅ are defined as above.

In one embodiment, the solvent used in the conversion of compound ofFormula 6 to a compound of Formula 7 is diethyl ether, or methylenechloride.

In another embodiment, the solvent used in the protection reaction ismethylene chloride.

In a further embodiment, PG is propoxy formyl, methanesulfonyl,4-nitro-benzoyl, ethoxy formyl, butoxy formyl, t-butoxy formyl,i-propoxy formyl or methoxy formyl.

In another embodiment, PG is methoxy formyl.

In another embodiment, a compound of Formula 7 is nitrated using amixture of sulfuric acid, nitric acid and methylene chloride.

In one embodiment, the nitro compound of Formula 8 is purified bycrystallization.

In a further embodiment, the nitro compound of Formula 8 is purified bycrystallization using hexane.

In another embodiment, the process further comprises the step ofcontacting a compound of Formula 4

with an aqueous acid to produce a compound of Formula 2.

In one embodiment, the compound of Formula 3 is a compound of Formula 40

In another embodiment, the process further comprises the step ofcontacting a compound of Formula 41

with methyl trimethylsilyl dimethylketene acetal (MTDA)

to produce a compound of Formula 42

In a further embodiment, the process comprises the step of reducing acompound of Formula 42 to produce a compound of Formula 40.

In one embodiment, the compound of Formula 3 is a compound of Formula 43

In a further embodiment, the process comprises the step of contacting acompound of

with methyl trimethlsilyl dimethylketene acetal (MTDA)

to produce a compound of Formula 45

In a further embodiment, the process comprises the step of reducing acompound of Formula 45 to produce a compound of Formula 43.

In another aspect, the invention provides a process for the preparationof a compound of Formula 2

comprising contacting a compound of Formula 4

with an aqueous acid, wherein

each X is independently a bond or is an optionally substituted C₁₋₆alkylidene chain wherein up to two methylene units of X are optionallyand independently replaced by —CO—, —CS—, —COCO—, —CONR′—, —CONR′NR′—,—CO₂—, —OCO—, —NR′CO₂—, —O—, —NR′CONR′—, —OCONR′—, —NR′NR′, —NR′NR′CO—,—NR′CO—, —S—, —SO, —SO₂—, —NR′—, —SO₂NR′—, NR′SO₂—, or —NR′SO₂NR′—;

each R^(x) is independently R′, halo, NO₂, CN, CF₃, or OCF₃;

y is an integer from 0-4; and

each R′ is independently selected from hydrogen or an optionallysubstituted group selected from a C₁₋₈ aliphatic group, a 3-8-memberedsaturated, partially unsaturated, or fully unsaturated monocyclic ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or an 8-12 membered saturated, partially unsaturated, or fullyunsaturated bicyclic ring system having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; or two occurrences of R′ aretaken together with the atom(s) to which they are bound to form anoptionally substituted 3-12 membered saturated, partially unsaturated,or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatomsindependently selected from N, O, or S.

In one embodiment of this aspect, the compound of Formula 4

was prepared by contacting a compound of Formula 50

with a compound of Formula 51

wherein R^(A), R^(B) and R^(C) can be C₁₋₆ alkyl.

In one embodiment of this aspect, the compound of Formula 50 and thecompound of Formula 50 are reacted at a temperature from about 100° C.to about 300° C. In another embodiment, the compound of Formula 50 andthe compound of Formula 50 are reacted at a temperature of about 100° C.In another embodiment, the compound of Formula 50 and the compound ofFormula 50 are reacted at a temperature of about 250° C. In one furtherembodiment, the compound of Formula 50 and the compound of Formula 50are reacted at a temperature of about 100° C., and then at a temperatureof about 250° C.

In one further embodiment of this aspect, y is 0.

In another aspect, the invention provides a process for the preparationof a compound of Formula 40

comprising the step of contacting a compound of Formula 41

with methyl trimethylsilyl dimethylketene acetal (MTDA)

to produce a compound of Formula 42

wherein

each R₂ is independently selected from hydrogen, CN, CF₃, halo, C₁₋₆straight or branched alkyl, 3-12 membered cycloaliphatic, phenyl, C₅₋₁₀heteroaryl or C₃₋₇ heterocyclic, wherein said heteroaryl or heterocyclichas up to 3 heteroatoms selected from O, S, or N, and each C₁₋₆ straightor branched alkyl, 3-12 membered cycloaliphatic, phenyl, C₅₋₁₀heteroaryl or C₃₋₇ heterocyclic is independently and optionallysubstituted with up to three substituents selected from —OR, —CF₃,—OCF₃, SR′, S(O)R′, SO₂R′, —SCF₃, halo, CN, —COOR′, —COR—,—O(CH₂)₂N(R′)(R′), —O(CH₂)N(R′)(R′), —CON(R′)(R′), —(CH₂)₂OR′,—(CH₂)OR′, CH₂CN, optionally substituted phenyl or phenoxy, —N(R′)(R′),—NR′C(O)OR′, —NR′C(O)R′, —(CH₂)₂N(R′)(R′), or —(CH₂)N(R′)(R′);

each R₅ is independently selected from hydrogen, —OH, NH₂, CN, CHF₂,NHR′, N(R′)₂, —NHC(O)R′, NHC(O)OR′, NHSO₂R′, —OR′, OC(O)OR′, OC(O)NHR′,OC(O)NR′₂, CH₂OH, CH₂N(R′)₂, C(O)OR′, SO₂NHR′, SO₂N(R′)₂, orCH₂NHC(O)OR′; and

each R′ is independently selected from hydrogen or an optionallysubstituted group selected from a C₁₋₈ aliphatic group, a 3-8-memberedsaturated, partially unsaturated, or fully unsaturated monocyclic ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or an 8-12 membered saturated, partially unsaturated, or fullyunsaturated bicyclic ring system having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; or two occurrences of R′ aretaken together with the atom(s) to which they are bound to form anoptionally substituted 3-12 membered saturated, partially unsaturated,or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatomsindependently selected from N, O, or S.

In one embodiment of this aspect, the process comprises the step ofreducing a compound of Formula 42 to produce a compound of Formula 40.

In another aspect, the invention provides a process for the preparationof a compound of Formula 43

comprising the step of contacting a compound having the Formula 44

with methyl trimethylsilyl dimethylketene acetal (MTDA)

to produce a compound of Formula 45

each R₂ is independently selected from hydrogen, CN, CF₃, halo, C₁₋₆straight or branched alkyl, 3-12 membered cycloaliphatic, phenyl, C₅₋₁₀heteroaryl or C₃₋₇ heterocyclic, wherein said heteroaryl or heterocyclichas up to 3 heteroatoms selected from O, S, or N, and each C₁₋₆ straightor branched alkyl, 3-12 membered cycloaliphatic, phenyl, C₅₋₁₀heteroaryl or C₃₋₇ heterocyclic is independently and optionallysubstituted with up to three substituents selected from —OR, —CF₃,—OCF₃, SR′, S(O)R′, SO₂R′, —SCF₃, halo, CN, —COOR′, —COR—,—O(CH₂)₂N(R′)(R′), —O(CH₂)N(R′)(R′), —CON(R′)(R′), —(CH₂)₂OR′,—(CH₂)OR′, CH₂CN, optionally substituted phenyl or phenoxy, —N(R′)(R′),—NR′C(O)OR′, —NR′C(O)R′, —(CH₂)₂N(R′)(R′), or —(CH₂)N(R′)(R′); and

each R′ is independently selected from hydrogen or an optionallysubstituted group selected from a C₁₋₈ aliphatic group, a 3-8-memberedsaturated, partially unsaturated, or fully unsaturated monocyclic ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or an 8-12 membered saturated, partially unsaturated, or fullyunsaturated bicyclic ring system having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; or two occurrences of R′ aretaken together with the atom(s) to which they are bound to form anoptionally substituted 3-12 membered saturated, partially unsaturated,or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatomsindependently selected from N, O, or S.

In one embodiment of this aspect, the process comprises the step ofreducing a compound of Formula 45 to produce a compound of Formula 43.

In some specific embodiments, a process for the preparation of compound27

comprises:

(a) reacting compound 26

with compound 13

in the presence of EDCI, HOBT and DIEA using DMF as the solvent, whereinthe reaction temperature is maintained between about 20° C. and 30° C.,and the reaction is allowed proceed for at least 70 hours, to producecompound 14

and(b) treating compound 14 with KOH in methanol.

In another specific embodiment, a process for the preparation ofcompound 28

comprises:

(a) reacting compound 26

with compound 20

in the presence of HATU and DIEA using acetonitrile as the solvent,wherein the reaction temperature is maintained between about 40° C. and50° C., and wherein the reaction is allowed proceed for at least 3 days,to produce compound 21

(b) treating compound 21 with NaOH in methanol.

In yet another specific embodiment, a process for the preparation ofcompound 34

comprises:

(a) reacting compound 26

with compound 32

in the presence of T3P® and pyridine using 2-methyl tetrahydrofuran asthe solvent, wherein the reaction temperature is maintained betweenabout 42° C. and about 3° C., and wherein the reaction is allowedproceed for at least 2 hours, to produce compound 33

and

(b) treating compound 33 with NaOMe/MeOH in 2-methyl tetrahydrofuran.

In another embodiment, the method also includes the step of forming aslurry of compound 34 in a mixture of acetonitrile and water, whereinthe solid form of compound 34 is converted to Compound 34.

In one embodiment, the ratio of acetonitrile to water is about 9:1 inthe slurry.

In another embodiment, the slurry is heated to a temperature betweenabout 73° C. and 83° C.

In another embodiment, compound 34 is in the slurry for at least about 3hours.

In a further embodiment, the process includes quenching the reactionmixture with 1N HCl; adding 0.1N HCl to the mixture, thereby creating abiphasic mixture; agitating the biphasic mixture; separating the organicphase from said biphasic mixture; filtering and removing solid matterfrom said organic phase; reducing the volume of the organic phase byapproximately 50% using distillation; performing thrice the steps of:adding acetonitrile to the organic phase until the volume of saidorganic phase increases by 100% and reducing the volume of the organicphase by approximately 50%; increasing the volume of the organic phaseby approximately 100% by adding acetonitrile and then adding water, toform a slurry wherein the final solvent ratio is 9:1 acetonitrile/water;heating said slurry to a temperature between about 73° C. and 83° C.;stirring said slurry for at least 5 hours; and cooling said slurry to atemperature between about −5° C. and 5° C.

In an alternative embodiment, the process includes quenching thereaction mixture with 1.2 N HCl; thereby creating a biphasic mixture;agitating said biphasic mixture; separating the organic phase from saidbiphasic mixture; adding 0.1N HCl to the organic layer thereby creatinga biphasic mixture; agitating said biphasic mixture; separating theorganic phase; filtering and removing solid matter from said organicphase; reducing the volume of the organic phase by approximately 50%using distillation; performing thrice the steps of: adding acetonitrileto the organic phase until the volume of said organic phase increases by100% and reducing the volume of the organic phase by approximately 50%;increasing the volume of the organic phase by approximately 100% byadding acetonitrile and then adding water, to form a slurry wherein thefinal solvent ratio is 9:1 acetonitrile/water; heating said slurry to atemperature between about 73° C. and 83° C.; stirring said slurry for atleast 5 hours; and cooling said slurry to a temperature between about20° C. and 25° C.; filtering and removing solid matter from said slurry;washing the solid matter with acetonitrile having a temperature ofbetween about 20° C. and 25° C. four times; and drying the solidmaterial under vacuum at a temperature of from 45° C. to about 55° C.

In one embodiment, the volume of 1N HCl used to quench the reaction isequal to 25% of the total volume of the original reaction mixture; thevolume of 0.1N HCl added to the reaction mixture is equal to 25% of thetotal volume of the original reaction mixture; and the distillationsteps are performed at reduced pressure wherein the temperature outsidethe reaction vessel is less than about 45° C. and the temperature of thereaction mixture is more than about 0° C.

In a further embodiment, the process includes forming a slurry ofcompound 34 in isopropyl acetate.

In one embodiment, the slurry is heated to reflux temperature.

In another embodiment, compound 34 is in the slurry for at least about 3hours.

In certain embodiments, the process for the preparation of Compound 34further comprises dissolving compound 34 in 2-methyltetrahydrofuran;adding 0.1N HCl to the solution, to creating a biphasic solution, whichis stirred. In another embodiment, the process further comprisesseparating the organic phase from the biphasic solution. In anotherembodiment, the process further comprises filtering and removing solidmatter from the organic phase. In another embodiment, the processfurther comprises reducing the volume of the organic phase byapproximately 50% using distillation. In another embodiment, the processfurther comprises performing thrice the procedure of: adding MeOAc,EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF), Et₂O or methyl-t-butylether (MTBE) to the organic phase until the volume of the organic phaseincreases by 100% and reducing the volume of the organic phase by 50%using distillation. In another embodiment, the process further comprisesadding MeOAc, EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF), Et₂O ormethyl-t-butyl ether (MTBE) to the organic phase until the volume of theorganic phase increases by 100%. In another embodiment, the processfurther comprises heating the organic phase to reflux temperature, andmaintaining said reflux temperature for a time at least about 5 hours.In another embodiment, the process further comprises cooling the organicphase to a temperature between about −5° C. and about 5° C. over a timeperiod of 4.5 hours to 5.5 hours.

In another embodiment, the process for the preparation of Compound 34further comprises crystallizing Compound 34, comprising seeding asaturated reaction mixture comprising Compound 34 in solution with atleast one crystal of substantially pure Compound 34.

In another embodiment, the invention provides a process for thepreparation of a compound of Formula 2

comprising hydrolyzing a compound of Formula 4

In a further embodiment, the compound of Formula 4 is hydrolyzed using ahydrolyzing agent in the presence of a solvent.

In some further embodiments, the hydrolyzing agent is HCl, H₂SO₄, H₃PO₄,Na₂CO₃, LiOH, KOH, or NaOH.

In some embodiments, the solvent used in the hydrolysis is H₂O,methanol, ethanol, isopropanol or t-butanol.

In still other embodiments, the invention provides a compound producedby any process described herein.

In a further embodiment, the invention provides a pharmaceuticalcomposition comprising a compound produced by any process describedherein.

In one aspect, the invention provides a process for the preparation ofCompound 27

comprising contacting Compound 34

with a biological composition.

In one embodiment of this aspect, the biological composition includes abiological organism selected from the group consisting of fungi,bacteria and archaea.

In one embodiment, the biological composition is fungi. In a furtherembodiment, the fungi is a single cell fungi. In another embodiment, thefungi is a multicell fungi.

In a further embodiment, the fungi is a multicell fungi selected fromthe group consisting of Absidia, Aspergillus, Beauveria, Botrytis,Cunninghamella, Cyathus, Gliocladium, Mortierella, Mucor, Phanerochaete,Stemphylium, Syncephalastrum and Verticillium.

In a further embodiment, the fungi is a multicell fungi selected fromthe group consisting of Absidia pseudocylindrospora, Aspergillusalliaceus, Aspergillus ochraceus, Beauveria bassiana, Cunninghamellablakesleeana, Cunninghamella echinulata, Mortierella isabellina,Mucorplumbeus, Phanerochaete chrysosporium, Syncephalastrum racemosumand Verticillium theobromae.

In another embodiment, the fungi is a single cell fungi selected fromthe group consisting of Candida, Debaryomyces, Geotrichum, Pichia,Rhodotorula, Saccharomyces, Sporobolomyces, Williopsis and Yarrowia.

In further embodiment, the fungi is a single cell fungi selected fromthe group consisting of Candida paripsilosis, Debaryomyces hansenii,Geotrichum candidum, Pichia methanolica, Pichia subpellicosa,Rhodotorula glutinis, Rhodotorula mucaliginosa, Saccharomycescerevisiae, Sporobolomyces salmonicolor, Williopsis saturnis andYarrowia lipolytica.

In another embodiment, the biological organism is an archaea. In afurther embodiment, the archaea is Pyrococcus. In still a furtherembodiment, the archaea is Pyrococcus furiosus.

In another embodiment, the biological organism is a bacteria.

In a further embodiment, the bacteria is selected from the groupconsisting of Lactobacillus, Pseudomonas, Rhodococcus and Streptomyces.

In a further embodiment, the bacteria is selected from the groupconsisting of Lactobacillus reuterii, Pseudomonas methanolica,Rhodococcus erythropolis, Streptomyces griseus, Streptomyces griseolus,Streptomyces platensis and Streptomyces rimosus.

In still a further embodiment, the biological composition includesStreptomyces rimosus, or a fragment thereof.

In one embodiment of this aspect, the biological composition includes asolvent. In a further embodiment, the solvent includes water. In still afurther embodiment, the solvent is a buffer. In still a furtherembodiment, the solvent is a potassium phosphate buffer having a pH ofabout 7.

In one aspect, the invention provides a process for the preparation ofCompound 28

comprising reacting Compound 34

with a biological composition.

In one embodiment of this aspect, the biological composition includes abiological organism selected from the group consisting of fungi,bacteria and archaea.

In one embodiment, the biological composition is fungi. In a furtherembodiment, the fungi is a single cell fungi. In another embodiment, thefungi is a multicell fungi.

In a further embodiment, the fungi is a multicell fungi selected fromthe group consisting of Absidia, Aspergillus, Beauveria, Botrytis,Cunninghamella, Cyathus, Gliocladium, Mortierella, Mucor, Phanerochaete,Stemphylium, Syncephalastrum and Verticillium.

In a further embodiment, the fungi is a multicell fungi selected fromthe group consisting of Absidia pseudocylindrospora, Aspergillusalliaceus, Aspergillus ochraceus, Beauveria bassiana, Cunninghamellablakesleeana, Cunninghamella echinulata, Mortierella isabellina,Mucorplumbeus, Phanerochaete chrysosporium, Syncephalastrum racemosumand Verticillium theobromae.

In another embodiment, the fungi is a single cell fungi selected fromthe group consisting of Candida, Debaryomyces, Geotrichum, Pichia,Rhodotorula, Saccharomyces, Sporobolomyces, Williopsis and Yarrowia.

In further embodiment, the fungi is a single cell fungi selected fromthe group consisting of Candida paripsilosis, Debaryomyces hansenii,Geotrichum candidum, Pichia methanolica, Pichia subpellicosa,Rhodotorula glutinis, Rhodotorula mucaliginosa, Saccharomycescerevisiae, Sporobolomyces salmonicolor, Williopsis saturnis andYarrowia lipolytica.

In another embodiment, the biological organism is an archaea. In afurther embodiment, the archaea is Pyrococcus. In still a furtherembodiment, the archaea is Pyrococcus furiosus.

In another embodiment, the biological organism is a bacteria.

In a further embodiment, the bacteria is selected from the groupconsisting of Lactobacillus, Pseudomonas, Rhodococcus and Streptomyces.

In a further embodiment, the bacteria is selected from the groupconsisting of Lactobacillus reuterii, Pseudomonas methanolica,Rhodococcus erythropolis, Streptomyces griseus, Streptomyces griseolus,Streptomyces platensis and Streptomyces rimosus.

In one embodiment of this aspect, the biological composition includesStreptomyces rimosus, or a fragment thereof.

In one embodiment of this aspect, the biological composition includes asolvent. In a further embodiment, the solvent includes water. In still afurther embodiment, the solvent is a buffer. In still a furtherembodiment, the solvent is a potassium phosphate buffer having a pH ofabout 7.

III. General Synthesis

Compounds of Formula 1 can be synthesized according to Scheme 1.

In Scheme 1, anilines of Formula 3, wherein R₂, R₄ and R₅ are optionallyand independently substituted with functional groups defined above, andwherein those functional groups optionally and independently bearprotecting groups thereon, are reacted with carboxylic acidintermediates of Formula 2 under coupling conditions. Derivatives ofFormula 1 that bear one or more protecting groups can then bedeprotected to provide unprotected derivatives of Formula 1.

The coupling reaction described in Scheme 1 can be achieved bydissolving the reactants in a suitable solvent, treating the resultingsolution with a suitable coupling reagent optionally in the presence ofa suitable base.

Anilines of Formula 3, wherein R₄ is a protected1-hydroxy-2-methylpropan-2-yl can be synthesized according to Scheme 2.

Alternatively, anilines of Formula 3, wherein R₄ is a protected1-hydroxy-2-methylpropan-2-yl can be synthesized according to Scheme 3.

Anilines of Formula 3, wherein R₄ and R₅ together with the phenyl ringto which they are attached form a 3,3-dimethylbenzofuran-2(3H)-one, canbe synthesized according to Scheme 4.

Alternatively, anilines of Formula 3, wherein R₄ and R₅ together withthe phenyl ring to which they are attached form a3,3-dimethylbenzofuran-2(3H)-one, can be synthesized according to Scheme5.

Anilines of Formula 3, wherein R₅ is a protected hydroxyl, can besynthesized according to Scheme 6.

Dihydroquinoline carboxylic acids of Formula 2 can be synthesizedaccording to Scheme 7, wherein the aniline derivative undergoesconjugate addition to EtOCH═C(COOEt)₂, followed by thermal rearrangementand hydrolysis.

IV. Uses and Methods of Use

Pharmaceutically Acceptable Compositions

In one aspect of the present invention, pharmaceutically acceptablecompositions are provided, wherein these compositions comprise any ofthe compounds as described herein, and optionally comprise apharmaceutically acceptable carrier, adjuvant or vehicle. In certainembodiments, these compositions optionally further comprise one or moreadditional therapeutic agents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative or a prodrug thereof. Accordingto the present invention, a pharmaceutically acceptable derivative or aprodrug includes, but is not limited to, pharmaceutically acceptablesalts, esters, salts of such esters, or any other adduct or derivativewhich upon administration to a patient in need thereof is capable ofproviding, directly or indirectly, a compound as otherwise describedherein, or a metabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, edisylate(ethanedisulfonate), ethanesulfonate, formate, fumarate, glucoheptonate,glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersable products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating, or lessening the severity of a condition, disease, or disorderimplicated by CFTR mutation. In certain embodiments, the presentinvention provides a method of treating a condition, disease, ordisorder implicated by a deficiency of the CFTR activity, the methodcomprising administering a composition comprising a compound of Formula1 to a subject, preferably a mammal, in need thereof.

In another aspect, the invention also provides a method of treating orlessening the severity of a disease in a patient comprisingadministering to said patient one of the compositions as defined herein,and said disease is selected from cystic fibrosis, asthma, smoke inducedCOPD, chronic bronchitis, rhinosinusitis, constipation, pancreatitis,pancreatic insufficiency, male infertility caused by congenitalbilateral absence of the vas deferens (CBAVD), mild pulmonary disease,idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA),liver disease, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington's, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease, Osteoporosis, Osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, and PrimaryCiliary Dyskinesia (PCD), a term for inherited disorders of thestructure and/or function of cilia, including PCD with situs inversus(also known as Kartagener syndrome), PCD without situs inversus andciliary aplasia.

In some embodiments, the method includes treating or lessening theseverity of cystic fibrosis in a patient comprising administering tosaid patient one of the compositions as defined herein. In certainembodiments, the patient possesses mutant forms of human CFTR. In otherembodiments, the patient possesses one or more of the followingmutations ΔF508, R117H, and G551D of human CFTR. In one embodiment, themethod includes treating or lessening the severity of cystic fibrosis ina patient possessing the ΔF508 mutation of human CFTR comprisingadministering to said patient one of the compositions as defined herein.In one embodiment, the method includes treating or lessening theseverity of cystic fibrosis in a patient possessing the G551D mutationof human CFTR comprising administering to said patient one of thecompositions as defined herein. In one embodiment, the method includestreating or lessening the severity of cystic fibrosis in a patientpossessing the ΔF508 mutation of human CFTR on at least one allelecomprising administering to said patient one of the compositions asdefined herein. In one embodiment, the method includes treating orlessening the severity of cystic fibrosis in a patient possessing theΔF508 mutation of human CFTR on both alleles comprising administering tosaid patient one of the compositions as defined herein. In oneembodiment, the method includes treating or lessening the severity ofcystic fibrosis in a patient possessing the G551D mutation of human CFTRon at least one allele comprising administering to said patient one ofthe compositions as defined herein. In one embodiment, the methodincludes treating or lessening the severity of cystic fibrosis in apatient possessing the G551D mutation of human CFTR on both allelescomprising administering to said patient one of the compositions asdefined herein.

In some embodiments, the method includes lessening the severity ofcystic fibrosis in a patient comprising administering to said patientone of the compositions as defined herein. In certain embodiments, thepatient possesses mutant forms of human CFTR. In other embodiments, thepatient possesses one or more of the following mutations ΔF508, R117H,and G551D of human CFTR. In one embodiment, the method includeslessening the severity of cystic fibrosis in a patient possessing theΔF508 mutation of human CFTR comprising administering to said patientone of the compositions as defined herein. In one embodiment, the methodincludes lessening the severity of cystic fibrosis in a patientpossessing the G551D mutation of human CFTR comprising administering tosaid patient one of the compositions as defined herein. In oneembodiment, the method includes lessening the severity of cysticfibrosis in a patient possessing the ΔF508 mutation of human CFTR on atleast one allele comprising administering to said patient one of thecompositions as defined herein. In one embodiment, the method includeslessening the severity of cystic fibrosis in a patient possessing theΔF508 mutation of human CFTR on both alleles comprising administering tosaid patient one of the compositions as defined herein. In oneembodiment, the method includes lessening the severity of cysticfibrosis in a patient possessing the G551D mutation of human CFTR on atleast one allele comprising administering to said patient one of thecompositions as defined herein. In one embodiment, the method includeslessening the severity of cystic fibrosis in a patient possessing theG551D mutation of human CFTR on both alleles comprising administering tosaid patient one of the compositions as defined herein.

In some aspects, the invention provides a method of treating orlessening the severity of Osteoporosis in a patient comprisingadministering to said patient compound of Formula 1 or apharmaceutically acceptable salt thereof.

In some embodiments, the method of treating or lessening the severity ofOsteoporosis in a patient comprises administering to said patientsubstantially amorphous compound of Formula 1 or a pharmaceuticallyacceptable salt thereof.

In still other embodiments, the method of treating or lessening theseverity of Osteoporosis in a patient comprises administering to saidpatient amorphous compound of Formula 1 or a pharmaceutically acceptablesalt thereof.

In certain embodiments, the method of treating or lessening the severityof Osteoporosis in a patient comprises administering to said patient apharmaceutical composition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of Osteopenia in a patient comprisingadministering to said patient compound of Formula 1 or apharmaceutically acceptable salt thereof.

In some embodiments, the method of treating or lessening the severity ofOsteopenia in a patient comprises administering to said patientsubstantially amorphous compound of Formula 1 or a pharmaceuticallyacceptable salt thereof.

In still other embodiments, the method of treating or lessening theseverity of Osteopenia in a patient comprises administering to saidpatient amorphous compound of Formula 1.

In certain embodiments, the method of treating or lessening the severityof Osteopenia in a patient comprises administering to said patient apharmaceutical composition as described herein.

In some aspects, the invention provides a method of bone healing and/orbone repair in a patient comprising administering to said patientcompound of Formula 1 or a pharmaceutically acceptable salt thereof.

In some embodiments, the method of bone healing and/or bone repair in apatient comprises administering to said patient substantially amorphouscompound of Formula 1 or a pharmaceutically acceptable salt thereof.

In still other embodiments, the method of bone healing and/or bonerepair in a patient comprises administering to said patient amorphouscompound of Formula 1 or a pharmaceutically acceptable salt thereof.

In certain embodiments, the method of bone healing and/or bone repair ina patient comprises administering to said patient a pharmaceuticalcomposition as described herein.

In some aspects, the invention provides a method of reducing boneresorption in a patient comprising administering to said patientcompound of Formula 1 or a pharmaceutically acceptable salt thereof.

In some embodiments, the method of reducing bone resorption in a patientcomprises administering to said patient substantially amorphous compoundof Formula 1 or a pharmaceutically acceptable salt thereof.

In still other embodiments, the method of reducing bone resorption in apatient comprises administering to said patient amorphous compound ofFormula 1 or a pharmaceutically acceptable salt thereof.

In some aspects, the invention provides a method of increasing bonedeposition in a patient comprising administering to said patientcompound of Formula 1 or a pharmaceutically acceptable salt thereof.

In some embodiments, the method of increasing bone deposition in apatient comprises administering to said patient substantially amorphouscompound of Formula 1 or a pharmaceutically acceptable salt thereof.

In still other embodiments, the method of increasing bone deposition ina patient comprises administering to said patient amorphous compound ofFormula 1 or a pharmaceutically acceptable salt thereof.

In certain embodiments, the method of increasing bone deposition in apatient comprises administering to said patient a pharmaceuticalcomposition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of COPD in a patient comprising administering tosaid patient compound of Formula 1 or a pharmaceutically acceptable saltthereof.

In some embodiments, the method of treating or lessening the severity ofCOPD in a patient comprises administering to said patient substantiallyamorphous compound of Formula 1 or a pharmaceutically acceptable saltthereof.

In still other embodiments, the method of treating or lessening theseverity of COPD in a patient comprises administering to said patientamorphous compound of Formula 1 or a pharmaceutically acceptable saltthereof.

In certain embodiments, the method of treating or lessening the severityof COPD in a patient comprises administering to said patient apharmaceutical composition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of smoke induced COPD in a patient comprisingadministering to said patient compound of Formula 1 or apharmaceutically acceptable salt thereof.

In some embodiments, the method of treating or lessening the severity ofsmoke induced COPD in a patient comprises administering to said patientsubstantially amorphous compound of Formula 1 or a pharmaceuticallyacceptable salt thereof.

In still other embodiments, the method of treating or lessening theseverity of smoke induced COPD in a patient comprises administering tosaid patient amorphous compound of Formula 1 or a pharmaceuticallyacceptable salt thereof.

In certain embodiments, the method of treating or lessening the severityof smoke induced COPD in a patient comprises administering to saidpatient a pharmaceutical composition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of chronic bronchitis in a patient comprisingadministering to said patient compound of Formula 1 or apharmaceutically acceptable salt thereof.

In some embodiments, the method of treating or lessening the severity ofchronic bronchitis in a patient comprises administering to said patientsubstantially amorphous compound of Formula 1 or a pharmaceuticallyacceptable salt thereof.

In still other embodiments, the method of treating or lessening theseverity of chronic bronchitis in a patient comprises administering tosaid patient amorphous compound of Formula 1 or a pharmaceuticallyacceptable salt thereof.

In certain embodiments, the method of treating or lessening the severityof chronic bronchitis in a patient comprises administering to saidpatient a pharmaceutical composition as described herein.

According to an alternative embodiment, the present invention provides amethod of treating cystic fibrosis comprising the step of administeringto said mammal an effective amount of a composition comprising acompound of the present invention.

According to the invention an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective fortreating or lessening the severity of one or more of the diseases,disorders or conditions as recited above.

Another aspect of the present invention provides a method ofadministering a pharmaceutical composition by orally administering to apatient at least once per day the composition comprising a compound ofFormula 1. In one embodiment, the method comprises administering apharmaceutical composition comprising a compound of Formula 1 every 24hours. In another embodiment, the method comprises administering apharmaceutical composition comprising a compound of Formula 1 every 12hours. In a further embodiment, the method comprises administering apharmaceutical composition comprising a compound of Formula 1 threetimes per day. In still a further embodiment, the method comprisesadministering a pharmaceutical composition comprising a compound ofFormula 1 every 4 hours.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of oneor more of the diseases, disorders or conditions as recited above.

In certain embodiments, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who exhibit residual CFTR activity in the apicalmembrane of respiratory and non-respiratory epithelia. The presence ofresidual CFTR activity at the epithelial surface can be readily detectedusing methods known in the art, e.g., standard electrophysiological,biochemical, or histochemical techniques. Such methods identify CFTRactivity using in vivo or ex vivo electrophysiological techniques,measurement of sweat or salivary CF concentrations, or ex vivobiochemical or histochemical techniques to monitor cell surface density.Using such methods, residual CFTR activity can be readily detected inpatients heterozygous or homozygous for a variety of differentmutations, including patients homozygous or heterozygous for the mostcommon mutation, ΔF508.

In another embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who have residual CFTR activity induced oraugmented using pharmacological methods or gene therapy. Such methodsincrease the amount of CFTR present at the cell surface, therebyinducing a hitherto absent CFTR activity in a patient or augmenting theexisting level of residual CFTR activity in a patient.

In one embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients within certain genotypes exhibiting residual CFTRactivity, e.g., class III mutations (impaired regulation or gating),class IV mutations (altered conductance), or class V mutations (reducedsynthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II, III, IV,and V cystic fibrosis Transmembrane Conductance Regulator Defects andOpportunities of Therapy; Current Opinion in Pulmonary Medicine6:521-529, 2000). Other patient genotypes that exhibit residual CFTRactivity include patients homozygous for one of these classes orheterozygous with any other class of mutations, including class Imutations, class II mutations, or a mutation that lacks classification.

In one embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients within certain clinical phenotypes, e.g., amoderate to mild clinical phenotype that typically correlates with theamount of residual CFTR activity in the apical membrane of epithelia.Such phenotypes include patients exhibiting pancreatic insufficiency orpatients diagnosed with idiopathic pancreatitis and congenital bilateralabsence of the vas deferens, or mild lung disease.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or patch), bucally, as an oral or nasal spray,or the like, depending on the severity of the infection being treated.In certain embodiments, the compounds of the invention may beadministered orally or parenterally at dosage levels of about 0.01 mg/kgto about 50 mg/kg and preferably from about 0.5 mg/kg to about 25 mg/kg,of subject body weight per day, one or more times a day, to obtain thedesired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are prepared by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The activity of a compound utilized in this invention as a modulator ofCFTR may be assayed according to methods described generally in the artand in the Examples herein.

It will also be appreciated that the compounds and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the compounds and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another agent used to treat the same disorder), orthey may achieve different effects (e.g., control of any adverseeffects). As used herein, additional therapeutic agents that arenormally administered to treat or prevent a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated.”

In one embodiment, the additional agent is selected from a mucolyticagent, bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than a compound of thepresent invention, or a nutritional agent.

In one embodiment, the additional agent is an antibiotic. Exemplaryantibiotics useful herein include tobramycin, including tobramycininhaled powder (TIP), azithromycin, aztreonam, including the aerosolizedform of aztreonam, amikacin, including liposomal formulations thereof,ciprofloxacin, including formulations thereof suitable foradministration by inhalation, levoflaxacin, including aerosolizedformulations thereof, and combinations of two antibiotics, e.g.,fosfomycin and tobramycin.

In another embodiment, the additional agent is a mucolyte. Exemplarymucolytes useful herein includes Pulmozyme®.

In another embodiment, the additional agent is a bronchodialator.Exemplary bronchodialtors include albuterol, metaprotenerol sulfate,pirbuterol acetate, salmeterol, or tetrabuline sulfate.

In another embodiment, the additional agent is effective in restoringlung airway surface liquid. Such agents improve the movement of salt inand out of cells, allowing mucus in the lung airway to be more hydratedand, therefore, cleared more easily. Exemplary such agents includehypertonic saline, denufosol tetrasodium([[(3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogenphosphate), or bronchitol (inhaled formulation of mannitol).

In another embodiment, the additional agent is an anti-inflammatoryagent, i.e., an agent that can reduce the inflammation in the lungs.Exemplary such agents useful herein include ibuprofen, docosahexanoicacid (DHA), sildenafil, inhaled glutathione, pioglitazone,hydroxychloroquine, or simavastatin.

In another embodiment, the additional agent is a CFTR modulator otherthan compound 1, i.e., an agent that has the effect of modulating CFTRactivity. Exemplary such agents include ataluren (“PTC124®”;3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid), sinapultide,lancovutide, depelestat (a human recombinant neutrophil elastaseinhibitor), cobiprostone (7-{(2R,4aR,5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta[b]pyran-5-yl}heptanoicacid), or (3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid. In anotherembodiment, the additional agent is(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.

In another embodiment, the additional agent is a nutritional agent.Exemplary such agents include pancrelipase (pancreating enzymereplacement), including Pancrease®, Pancreacarb®, Ultrase®, or Creon®,Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione inhalation.In one embodiment, the additional nutritional agent is pancrelipase.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating an implantable medical device, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present invention includes an implantable device coated witha composition comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

Another aspect of the invention relates to modulating CFTR activity in abiological sample or a patient (e.g., in vitro or in vivo), which methodcomprises administering to the patient, or contacting said biologicalsample with a compound of Formula 1 or a composition comprising saidcompound. The term “biological sample”, as used herein, includes,without limitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Modulation of CFTR in a biological sample is useful for a variety ofpurposes that are known to one of skill in the art. Examples of suchpurposes include, but are not limited to, the study of CFTR inbiological and pathological phenomena; and the comparative evaluation ofnew modulators of CFTR.

In yet another embodiment, a method of modulating activity of an anionchannel in vitro or in vivo, is provided comprising the step ofcontacting said channel with a compound of Formula 1. In embodiments,the anion channel is a chloride channel or a bicarbonate channel. Inother embodiments, the anion channel is a chloride channel.

According to an alternative embodiment, the present invention provides amethod of increasing the number of functional CFTR in a membrane of acell, comprising the step of contacting said cell with a compound ofFormula 1.

According to another embodiment, the activity of the CFTR is measured bymeasuring the transmembrane voltage potential. Means for measuring thevoltage potential across a membrane in the biological sample may employany of the known methods in the art, such as optical membrane potentialassay or other electrophysiological methods.

The optical membrane potential assay utilizes voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells.” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R.Y. Tsien (1997); “Improved indicators of cell membrane potential thatuse fluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission can be monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

In one embodiment, the present invention provides a method of modulatingCFTR activity in a biological sample comprising the step of contactingsaid biological sample with a compound of Formula 1, or apharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R₄ and Yare defined as above.

In one embodiment, the present invention provides a method of modulatingCFTR activity in a biological sample comprising the step of contactingsaid biological sample with a compound, produced via the processesdescribed herein, of the structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention provides a method of modulatingCFTR activity in a biological sample comprising the step of contactingsaid biological sample with a compound, produced via the processesdescribed herein, of the structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention provides a method of modulatingCFTR activity in a biological sample comprising the step of contactingsaid biological sample with a compound, produced via the processesdescribed herein, of the structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention provides a method of treatingor lessening the severity of a disease in a patient comprisingadministering to said patient an effective amount of a compound ofFormula 1, or a pharmaceutically acceptable salt thereof, wherein R₁,R₂, R₃, R₄ and Y are defined as above, and said disease is selected fromcystic fibrosis, asthma, smoke induced COPD, chronic bronchitis,rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency,male infertility caused by congenital bilateral absence of the vasdeferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,allergic bronchopulmonary aspergillosis (ABPA), liver disease,hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease.

In one embodiment, the method includes treating or lessening theseverity of a disease in a patient by administering to said patient aneffective amount of a compound, produced via the processes describedherein, having the structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the method includes treating or lessening theseverity of a disease in a patient by administering to said patient aneffective amount of a compound, produced via the processes describedherein, having the structure:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the method includes treating or lessening theseverity of a disease in a patient by administering to said patient aneffective amount of a compound, produced via the processes describedherein, having the structure:

or a pharmaceutically acceptable salt thereof.

In another aspect the present invention provides a kit for use inmeasuring the activity of CFTR or a fragment thereof in a biologicalsample in vitro or in vivo comprising (i) a composition comprising acompound of Formula 1 or any of the above embodiments; and (ii)instructions for a) contacting the composition with the biologicalsample and b) measuring activity of said CFTR or a fragment thereof.

In one embodiment, the kit further comprises instructions for a)contacting an additional composition with the biological sample; b)measuring the activity of said CFTR or a fragment thereof in thepresence of said additional compound, and c) comparing the activity ofthe CFTR in the presence of the additional compound with the density ofthe CFTR in the presence of a composition of Formula 1.

In embodiments, the kit is used to measure the density of CFTR.

In one embodiment, the kit includes a composition comprising a compound,produced via the processes described herein, having the structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the kit includes a composition comprising a compound,produced via the processes described herein, having the structure:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the kit includes a composition comprising acompound, produced via the processes described herein, having thestructure:

or a pharmaceutically acceptable salt thereof.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

V. Examples Preparation 1: Total Synthesis of4-oxo-1,4-dihydroquinoline-3-carboxylic acid (26)

Procedure for the preparation of ethyl4-oxo-1,4-dihydroquinoline-3-carboxylate (25)

Compound 23 (4.77 g, 47.7 mmol) was added dropwise to compound 22 (10 g,46.3 mmol) with subsurface N₂ flow to drive out ethanol below 30° C. for0.5 hours. The solution was then heated to 100-110° C. and stirred for2.5 hours. After cooling the mixture to below 60° C., diphenyl ether wasadded. The resulting solution was added dropwise to diphenyl ether thathad been heated to 228-232° C. for 1.5 hours with subsurface N₂ flow todrive out ethanol. The mixture was stirred at 228-232° C. for another 2hours, cooled to below 100° C. and then heptane was added to precipitatethe product. The resulting slurry was stirred at 30° C. for 0.5 hours.The solids were then filtrated, and the cake was washed with heptane anddried in vacuo to give compound 25 as brown solid. ¹H NMR (DMSO-d₆; 400MHz) δ 12.25 (s), δ 8.49 (d), δ 8.10 (m), δ 7.64 (m), δ 7.55 (m), δ 7.34(m), δ 4.16 (q), δ 1.23 (t).

Procedure for the preparation of 4-oxo-1,4-dihydroquinoline-3-carboxylicacid (26)

Method 1

Compound 25 (1.0 eq) was suspended in a solution of HCl (10.0 eq) andH₂O (11.6 vol). The slurry was heated to 85-90° C., although alternativetemperatures are also suitable for this hydrolysis step. For example,the hydrolysis can alternatively be performed at a temperature of fromabout 75 to about 100° C. In some instances, the hydrolysis is performedat a temperature of from about 80 to about 95° C. In others, thehydrolysis step is performed at a temperature of from about 82 to about93° C. (e.g., from about 82.5 to about 92.5° C. or from about 86 toabout 89° C.). After stirring at 85-90° C. for approximately 6.5 hours,the reaction was sampled for reaction completion. Stirring may beperformed under any of the temperatures suited for the hydrolysis. Thesolution was then cooled to 20-25° C. and filtered. The reactor/cake wasrinsed with H₂O (2 vol×2). The cake was then washed with 2 vol H₂O untilthe pH≧3.0. The cake was then dried under vacuum at 60° C. to givecompound 26.

Method 2

Compound 25 (11.3 g, 52 mmol) was added to a mixture of 10% NaOH (aq)(10 mL) and ethanol (100 mL). The solution was heated to reflux for 16hours, cooled to 20-25° C. and then the pH was adjusted to 2-3 with 8%HCl. The mixture was then stirred for 0.5 hours and filtered. The cakewas washed with water (50 mL) and then dried in vacuo to give compound26 as a brown solid. ¹H NMR (DMSO-d₆; 400 MHz) δ 15.33 (s), δ 13.39 (s),δ 8.87 (s), δ 8.26 (m), δ 7.87 (m), δ 7.80 (m), δ 7.56 (m).

Example 1: Total synthesis ofN-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(27)

The overall scheme of the synthesis of compound 27 is shown below,followed by the procedure for the synthesis of each syntheticintermediate.

Procedure for the preparation of 2-hydroxy-5-tert-butylbenzaldehyde (2)

To a stirred solution of compound 1 (700 g, 4.66 mol) in CH₃CN (7.0 L)was added MgCl₂ (887 g, 9.32 mol), Para-Formaldehyde (1190 g) and TEA(2.5 L, 17.9 mol) under N₂. The mixture was heated to reflux for 5hours. After cooling to room temperature, 2 L ice water was added to themixture, followed by 6 L of 3 M HCl (aq). The suspension was leftstirring until the solution became clear. The organic layer wasseparated and the aqueous layer was extracted with MTBE (3 L×3). Theorganic layers were combined and concentrated to dryness. The residuewas dissolved in MTBE (4000 mL), washed with water (1000 mL×2) and brine(1000 mL), dried over anhydrous Na₂SO₄, filtered, then concentrated togive compound 2 as a light-yellow solid which was used in the nextreaction without further drying or purification. 1H NMR (CDCl₃; 400 MHz)δ 10.86 (s), δ 9.89 (s), δ 7.59 (m), δ 7.51 (d), δ 6.94 (d), δ 10.61(s).

Procedure for the preparation of 2-(benzyloxy)-5-tert-butylbenzaldehyde(3)

To a stirred solution of compound 2 (614.5 g, 3.33 mol) in DMF (3.5 L)was added K₂CO₃ (953 g, 6.90 mol) and benzyl chloride (480 g, 3.80 mol).The mixture was heated to 90° C. and left stirring for 3 hours. Thesuspension was cooled to room temperature, then MTBE (2 L) was added,followed by water (12 L). The mixture was then stirred for 10 minutesand the aqueous layer was separated and extracted with MTBE (2 L×3). Theorganic layers were combined and washed with water (2 L×2) and brine(1.5 L×1) and concentrated to give compound 3 as a light-yellow solid.¹H NMR (DMSO-d₆; 400 MHz) δ 10.42 (s), δ 7.71 (m), δ 7.51 (m), δ 7.43(m), δ 7.35 (m), δ 7.24 (m), δ 5.27 (s), δ 1.26 (s).

Procedure for the preparation of 2-(benzyloxy)-5-tert-butylbenzylalcohol (4)

To a stirred suspension of compound 3 (974 g, 3.63 mol) in MeOH (4000mL) was slowly added NaBH₄ (121 g, 3.20 mol) at 0-20° C. The solutionwas left stirring at 15° C. for 3 hours, and then cooled to 0° C. 2N HCl(aq) (1300 mL) was added dropwise at below 20° C. The solution was thenfiltered and evaporated to dryness, and the residue was dissolved inMTBE (5 L). The solution was then washed with water (2 L×2) and brine(1.5 L×1). Evaporation of the solvent gave compound 4 as a light-yellowsolid which was used in the next reaction without further purification.¹H NMR (DMSO-d₆; 400 MHz) δ 7.40 (m), δ 7.32 (m), δ 7.17 (m), δ 6.91(m), δ 5.09 (s), δ 5.00 (t), δ 4.56 (d), δ 1.26 (s).

Procedure for the preparation of 2-(benzyloxy)-5-tert-butylbenzylchloride (5)

To a stirred solution of compound 4 (963 g, 3.56 mol) in anhydrous DCM(2000 mL) was added slowly SOCl₂ (535 g, 4.5 mol) at 0° C. The mixturewas stirred at 20° C. for 2 hours, then concentrated in vacuo to givecompound 5 as an oil, which was used in the next reaction withoutfurther drying or purification.

Procedure for the preparation of 2-(benzyloxy)-5-tert-butylbenzylnitrile (6)

To a stirred solution of compound 5 (1045 g, 3.54 mol) in anhydrous DMF(1000 mL) was added KCN (733 g, 11.3 mol). The mixture was stirred at35° C. for 24 hours, then poured into water (10 L). Ethyl acetate (4 L)was added and the mixture was stirred for 30 minutes. The organic layerwas then separated and the aqueous layer was extracted with ethylacetate (3000 mL×2). The organic layers were combined and washed withwater (4 L×2) and brine (3 L×1), then concentrated in vacuo to givecompound 6 as a yellow solid. ¹H NMR (DMSO-d₆; 400 MHz) δ 7.51 (m), δ7.37 (m), 7.02 (d), δ 5.17 (s), δ 3.88 (s), 1.26 (s).

Procedure for the preparation of2-(2-(benzyloxy)-5-tert-butylphenyl)-2-methylpropanenitrile (7)

To a stirred suspension of NaH (86 g, 2.15 mol, 60% in mineral oil) inDMF (1000 mL) was added dropwise a solution of compound 6 (100.0 g,0.358 mol) in DMF (500 mL) at 20° C. After stirring for 30 minutes, MeI(205 g, 1.44 mol) in DMF (500 mL) was added dropwise at below 30° C.during a period of 2 hours. The suspension was stirred for 1.5 hours at25-30° C., then ice (100 g) was added slowly until no gas was generated.The pH was adjusted to approximately 7 by the slow addition of 2N HCl.The mixture was diluted with water (4 L) and MTBE (2 L). The organiclayer was separated and the aqueous layer was extracted with MTBE (500mL×2). The combined organic layers were washed with water and brine,dried over Na₂SO₄, filtered, and then concentrated in vacuo to givecompound 7 as a white solid. ¹H NMR (DMSO-d₆; 400 MHz) δ 7.56 (m), δ7.40 (m), δ 7.34 (m), δ 7.10 (d), δ 5.21 (s), δ 1.73 (s), δ 1.27 (s).

Procedure for the preparation of2-(2-(benzyloxy)-5-tert-butylphenyl)-2-methylpropanal (8)

To a stirred solution of compound 7 (20 g, 0.065 mol) in toluene (300mL), was added drop wise DIBAH (80 mL, 1 M in toluene) at about −60 to−50° C. After stirring for 2 hours, 6 N HCl (300 mL) was added to thereaction mixture and stirring was continued for 30 minutes. The organiclayer was then separated, washed with 2 N HCl followed by a NaHCO₃solution, then a brine solution, dried over Na₂SO₄ and concentrated invacuo to afford the compound 8 as an oil. The product was used in thenext reaction without further purification. ¹H NMR (CDCl₃; 400 MHz) δ9.61 (s), δ 7.36 (m), δ 7.25 (m), δ 6.87 (m), δ 5.06 (m), δ 1.43 (s), δ1.33 (s).

Procedure for the preparation of2-(2-(benzyloxy)-5-tert-butylphenyl)-2-methylpropan-1-ol (9)

To a stirred solution of compound 8 (9.21 g, 0.030 mol) in MeOH (150 mL)was added slowly NaBH₄ (2.3 g, 0.061 mol) at 0° C. After the mixture wasstirred at 20° C. for 3 hours, 12 mL of 6 N HCl was added, and themixture was stirred for an additional 30 minutes. The solution was thenconcentrated to about one-quarter of the original volume and extractedwith EtOAc. The organic layer was separated and washed with water andbrine, dried with Na₂SO₄, filtered, and then concentrated in vacuo toafford compound 9 as a white solid. ¹H NMR (DMSO-d₆; 400 MHz) δ 7.47(m), δ 7.42 (m), δ 7.34 (m), δ 7.28 (m), δ 7.16 (m), δ 6.94 (m), δ 5.08(s), δ 4.45 (t), δ 3.64 (d), δ 1.28 (s), δ 1.25 (s).

Procedure for the preparation of2-(2-hydroxy-5-tert-butylphenyl)-2-methylpropan-1-ol (10)

Pd(OH)₂ (1 g) and compound 9 (9.26 g, 0.030 mol) in MeOH (200 mL) werestirred under hydrogen at 20-30 psi pressure for 16-18 hours. Themixture was then filtered through Celite®, and the filtrate wasconcentrated to give compound 10 as a white solid. ¹H NMR (DMSO-d₆; 400MHz) δ 9.16 (s), δ 7.16 (d), δ 7.00 (m), δ 6.65 (m), δ 4.71 (t), δ 3.62(d), δ 1.27 (s), δ 1.22 (s).

Procedure for the preparation of1-((methylcaroboxy)oxy)-2-(1-((methylcaroboxy)oxy)-2-methylpropan-2-yl)-4-tert-butylbenzene (11)

To a stirred solution of compound 10 (23.2 g, 0.10 mol), DMAP (1.44 g)and DIEA (72.8 g, 0.56 mol) in anhydrous DCM (720 mL) was added dropwisemethyl chloroformate (43.5 g, 0.46 mol) in DCM (160 mL) at 0° C. Afterthe mixture was stirred at 20° C. for 16 hours, it was washed withwater, 1 N HCl and brine, dried with MgSO₄ and concentrated in vacuo.The residue was purified using column chromatography on silica gel (1:20EtOAc:Petroleum ether) to give compound 11 as a white solid. ¹H NMR(DMSO-d₆; 400 MHz) δ 7.32 (m), δ 7.10 (d), δ 4.26 (s), δ 3.84 (s), δ3.64 (s), δ 1.31 (s), δ 1.28 (s).

Procedure for preparation ofl-((methylcaroboxy)oxy)-2-(1-((methylcaroboxy)oxy)-2-methylpropan-2-yl)-4-tert-butyl-5-nitrobenzene (12)

To a stirred solution of compound 11 (32 g, 0.095 mol) in DCM (550 mL)was added dropwise 98% H₂SO₄ (43 g, 0.43 mol) at 0° C. After stirringfor 20 minutes at 0° C., 65% HNO₃ (16.2 g, 0.17 mol) was added to themixture dropwise at 0° C. The mixture was then stirred at 1-10° C. for 4hours and then ice-water (200 mL) was added. The aqueous layer wasseparated and extracted with DCM (200 mL×3) and the combined organiclayers were washed with water (aq), NaHCO₃ and brine, then dried withMgSO₄ and concentrated in vacuo. The residue was purified via columnchromatography on silica gel (1:20 EtOAc:Petroleum ether) to affordcrude compound 12 as an oil.

Procedure for the preparation of2-tert-butyl-5-((methylcaroboxy)oxy)-4-(1-((methylcaroboxy)oxy)-2-methylpropan-2-yl)aniline (13)

Pd/C (2.6 g) and compound 12 (14 g, crude) were stirred in MeOH (420 mL)at room temperature under hydrogen at 20-30 psi pressure for 16-18hours. Then the mixture was filtered with Kieselguhr®, and the filtratewas concentrated in vacuo. The residue was purified via columnchromatography on silica gel (1:10 EtOAc:Petroleum ether) to givecompound 13 as a gray solid. ¹H NMR (CDCl₃; 400 MHz) δ 7.26 (s), δ 7.19(s), δ 4.26 (s), δ 3.89 (s), δ 3.74 (s), δ 1.40 (s), δ 1.35 (s).

Procedure for the preparation ofN-(2-tert-butyl-5-((methylcaroboxy)oxy)-4-(1-((methylcaroboxy)oxy)-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(14)

To a stirred solution of compound 26 (5.0 g, 0.026 mol) in anhydrous DMF(120 mL) was added EDCI (5.6 g, 0.029 mol), HOBT (3.8 g, 0.028 mol) andDIEA (6.6 g, 0.051 mol) at 0° C. After stirring for 1 hour, the mixturewas added dropwise a solution of compound 13 (3.0 g, 0.008 mol) in DCM(30 ml) at 0° C. The mixture was stirred at 25° C. for 72 hours, andthen was concentrated in vacuo. The residue was dissolved in EtOAc (225mL) and washed with water (120 mL×1), 1N HCl (120 mL) and brine, driedwith Na₂SO₄ and concentrated in vacuo. The residue was purified viacolumn chromatography on silica gel (1:1 EtOAc:Petroleum ether) to givecompound 14 as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 12.34 (s, 1H),11.58 (s, 1H), 9.07 (s, 1H), 8.42 (d, 1H), 7.66 (s, 1H), 7.51 (s, 1H),7.47 (s, 1H), 7.39 (s, 1H), 6.72 (s, 1H), 4.34 (s, 2H), 3.82 (s, 3H),3.74 (s, 3H), 1.41 (s, 9H), 1.40 (s, 6H).

Procedure for the preparation ofN-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(27)

To a stirred solution of KOH (1.2 g, 0.02 mol) in MeOH (80 mL) was addedcompound 14 (1.9 g, 0.0036 mol) at 0° C. After stirring for 2-3 hours at5-15° C., the mixture was concentrated to dryness. The residue was thentriturated in water (10 mL), filtered, washed with DCM and dried invacuo for 24 hours to give compound 27 as a white solid. ¹H NMR(DMSO-d₆; 400 MHz) δ 12.77 (s), δ 8.86 (s), δ 8.20 (d), δ 7.55 (d), δ7.42 (t), δ 7.16 (q), δ 7.02 (s), δ 6.85 (m), δ 3.55 (s), δ 1.55 (s), δ1.35 (s), δ 1.27 (s). MS Found (M+H) 409.2.

Example 2: Alternative Total Synthesis ofN-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(27)

Procedure for the preparation of methyl2-(5-tert-butyl-2-hydroxy-4-nitrophenyl)-2-methylpropanoate (38)

A mixture of 2-bromo-4-tert-butyl-5-nitrophenol (15.00 g, 54.72 mmol),bis(tri-tert-butylphospine)palladium(0) (1.422 g, 2.783 mmol), zincfluoride (2.82 g, 27.27 mmol), methyl trimethylsilyl dimethylketeneacetal (MTDA) (19.35 g, 111.0 mmol), and dimethylformamide (150 mL) washeated at 70° C. for 18 h. The mixture was cooled to room temperatureand diluted with water. After stirring for one hour, the aqueous phasewas extracted with MTBE. The organic layer was dried in vacuo to affordthe crude product as a brown solid. Purification of the product wasaccomplished by trituration in n-heptane. ¹H-NMR (400 MHZ, DMSO-d6) δ10.38 (s, 1H); 7.37 (s, 1H); 6.79 (s, 1H); 3.54 (s, 3H); 1.45 (s, 6H);1.32 (s, 9H)

Procedure for the preparation of4-tert-butyl-2-(1-hydroxy-2-methylpropan-2-yl)-5-nitrophenol (39)

A 1M solution of lithium aluminum hydride in THF (11.80 mL, 11.80 mmol)was added to a solution of methyl2-(5-tert-butyl-2-hydroxy-4-nitrophenyl)-2-methylpropanoate (5.36 g,18.15 mmol) in THF (50 mL). The mixture was stirred at ambienttemperature for 3 h, and then diluted with methanol. The mixture wasacidified with 1N HCl (pH 1-2) and the aqueous phase was extracted withMTBE. The organic phase was dried in vacuo to afford4-tert-butyl-2-(1-hydroxy-2-methylpropan-2-yl)-5-nitrophenol which wasused without further purification in the next step. ¹H-NMR (400 MHZ,DMSO-d6) δ 10.12 (s, 1H); 7.37 (s, 1H); 6.80 (s, 1H); 4.77 (s, 1H);3.69-3.65 (m, 2H); 1.30 (s, 9H); 1.29 (s, 6H)

Procedure for the preparation of4-tert-butyl-2-(2-methoxycarbonyloxy-1,1-dimethyl-ethyl)-5-nitro-phenyl]methylcarbonate (12)

To a solution of4-tert-butyl-2-(1-hydroxy-2-methylpropan-2-yl)-5-nitrophenol (1.92 g,7.18 mmol), triethylamine (1.745 g, 17.24 mmol), anddimethylaminopyridine (87.74 mg, 0.718 mmol) in dichloromethane (30 mL)at 0° C. was slowly charged methylchloroformate (2.376 g, 25.14 mmol),keeping the temperature below 5° C. After the addition, the mixture wasallowed to warm to ambient temperature and was stirred until HPLC showedcomplete conversion of the starting material (2-8 h). The reactionmixture was diluted with water and acidified with 1N HCl (pH 1-2). Theaqueous phase was extracted with DCM and the combined organics dried invacuo. The crude amber semi-solid was re-crystallized from methanol anddichloromethane to give the title compound as a yellow crystallinesolid. ¹H-NMR (400 MHZ, DMSO-d6) δ 7.67 (s, 1H); 7.52 (s, 1H); 4.30 (s,2H); 3.86 (s, 3H); 3.64 (s, 3H); 1.35 (s, 9H); 1.35 (s, 6H)

Procedure for the preparation of5-amino-4-tert-butyl-2-(2-methoxycarbonyloxy-1,1-dimethyl-ethyl)phenyl]methylcarbonate (13)

A mixture of[4-tert-butyl-2-(2-methoxycarbonyloxy-1,1-dimethyl-ethyl)-5-nitro-phenyl]methylcarbonate (1.27 g, 3.313 mmol) and Pd/C (75 mg, 0.035 mmol) in methanol(50 mL) was purged with nitrogen. After purging the flask with hydrogen,the mixture was hydrogenated for 18 hours at ambient temperature andpressure. The solution was filtered through Celite® and dried in vacuoto obtain the product as a solid. ¹H-NMR (400 MHZ, DMSO-d6) δ 6.99 (s,1H); 6.39 (s, 1H); 4.92 (s, 2H); 4.13 (s, 2H); 3.82 (s, 3H); 3.65 (s,3H); 1.32 (s, 9H); 1.23 (s, 6H)

Procedure for the preparation ofN-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(27)

To a mixture of[5-amino-4-tert-butyl-2-(2-methoxycarbonyloxy-1,1-dimethyl-ethyl)phenyl]methylcarbonate (103 mg, 0.29 mmol), 4-oxo-1,4-dihydroquinoline-3-carboxylicacid (50 mg, 0.26 mmol), and pyridine (42 mg, 0.53 mmol) in 2-MeTHF (3.0mL) was charged T3P as a 50 wt % solution in 2-MeTHF (286 mg, 0.45mmol). The mixture was heated to 50° C. for 18 h. After cooling toambient temperature, the mixture was diluted with water. The organicphase was separated and again washed with water. Sodium methoxide (39mg, 0.72 mmol) was charged to the organic phase and the solution stirredfor 2 hours. The reaction was quenched with 1N HCl, and after separatingthe phases, the organic phase was washed with 0.1N HCl. The organicphase was than dried in vacuo to yield Compound 27 as a solid. The¹H-NMR spectrum was consistent with that reported above.

Example 3: Total Synthesis of2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phenyl)-2-methylpropanoicacid (28)

Procedure for the preparation of2-(5-tert-butyl-2-hydroxyphenyl)-2-methylpropanenitrile (15)

Pd (OH)₂/C (2.0 g) and compound 7 (20.0 g, 0.104 mol) were stirred inMeOH (150 mL) at room temperature under hydrogen at 10 psi pressure for16-18 hours. The mixture was then filtered through a pad of Celite®, andthe filtrate was concentrated to give compound 15, which was used in thenext reaction without further purification. ¹H NMR (DMSO-d₆; 400 MHz) δ9.83 (s), δ 7.24 (s), δ 7.18 (m), δ 6.80 (m), δ 1.71 (s), δ 1.24 (s).

Procedure for the preparation of4-tert-butyl-2-(2-cyanopropan-2-yl)phenyl methyl carbonate (16)

To a stirred mixture of compound 15 (126.6 g, 0.564 mol), DMAP (6.0 g)and DIEA (188 g, 1.46 mol) in anhydrous DCM (1500 mL) was added dropwisemethyl chloroformate (110 g, 1.17 mol) in anhydrous DCM (300 mL) at 0°C. within 2 hours. After stirring for 12 hours at 0° C., ice-water (1.5L) was added and the mixture was stirred at 0° C. for 30 minutes. Theorganic layer was separated and washed with 1 N HCl, water, and brine.The DCM solution was dried over MgSO₄ and concentrated in vacuo to givecompound 16 as a yellow solid. ¹H NMR (DMSO-d₆; 400 MHz) a 7.47 (m), δ7.39 (d), δ 7.24 (d), δ 3.84 (s), δ 1.71 (s), δ 1.30 (s).

Procedure for the preparation of2-(1-amino-2-methyl-1-oxopropan-2-yl)-4-tert-butyl-5-nitrophenyl methylcarbonate (17)

To a stirred mixture of compound 16 (10.0 g, 36.3 mmol) and KNO₃ (5.51g, 54.5 mmol) in DCM (1000 mL) was added dropwise 98% H₂SO₄ (145.4 g,1.45 mol) at 0° C. The mixture was stirred at 30° C. for 4 days. TheH₂SO₄ layer was then separated and poured into ice-water (50 g) and thenextracted with DCM (100 mL×3). The combined organic layers were washedwith water, aqueous NaHCO₃ solution and brine, then dried over MgSO₄ andconcentrated in vacuo. The residue was purified via columnchromatography on silica gel (Petroleum ether/EtOAc 20:1→10:1→5:1→3:1)to give compound 17 as a yellow solid. ¹H NMR (CDCl₃; 400 MHz) δ 8.05(s), δ 7.74 (s), δ 7.61 (s), δ 7.32 (s), δ 5.32 (s), δ 3.91 (s), δ 3.92(s), δ 1.62 (s), δ 1.59 (s), δ 1.42 (s), δ 1.38 (s).

Procedure for the preparation of2-(5-tert-butyl-2-hydroxy-4-nitrophenyl)-2-methylpropanoic acid (18)

To a mixture of compound 17 (7.3 g, 21.6 mmol) in methanol (180 mL) wasadded water (18 mL) and NaOH (8.64 g, 216 mmol). The solution was heatedand maintained at reflux for 3 days. The solvent was evaporated in vacuoand the residue was dissolved in 140 mL of water. Then the solution wasacidified to pH 2 by the addition of 2N HCl. The aqueous phase wasextracted with ethyl acetate (100 mL×3), and the combined organic phaseswere washed with water and brine, dried over anhydrous Na₂SO₄ and thenconcentrated to give compound 18 as a yellow solid, which was used inthe next reaction without further purification.

Procedure for the preparation of5-tert-butyl-3,3-dimethyl-6-nitrobenzofuran-2(3H)-one (19)

To a solution of compound 18 (7.10 g, 25.2 mmol) in 710 mL of anhydrousTHF was added EDCI (14.5 g, 75.6 mmol). The resulting suspension wasleft stirring at 30° C. overnight. The precipitate was filtered andthoroughly washed with DCM. The filtrate was concentrated to dryness andthe residue was dissolved in DCM (100 mL). The solution was washed withwater (50 mL×2) and brine (50 mL×1). The DCM layer was then dried overanhydrous Na₂SO₄ and concentrated to give the crude product, which waspurified via column chromatography on silica gel (Petroleum ether/EtOAc200:1-100:1-50:1) to give compound 19 as a white solid. ¹H NMR (CDCl₃;400 MHz) δ 7.36 (s), δ 7.10 (s), δ 1.53 (s), δ 1.41 (s).

Procedure for the preparation of6-amino-5-tert-butyl-3,3-dimethylbenzofuran-2(3H)-one (20)

Pd/C (1.50 g) and compound 19 (3.00 g, 1.14 mmol) were suspended in THF(1500 mL) at 25° C. under hydrogen at 30 psi for 4 hours. The mixturewas then filtered through a pad of Celite®, and the filtrate wasconcentrated in vacuo to give compound 20 as a white solid. ¹H NMR(DMSO-d₆; 400 MHz) δ 7.05 (s), δ 6.49 (s), δ 5.01 (s), δ 1.35 (s), δ1.33 (s).

Procedure for the preparation ofN-(5-tert-butyl-3,3-dimethyl-2-oxo-2,3-dihydrobenzofuran-6-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(21)

A suspension of HATU (17.6 g, 46.3 mol) and compound 26 (8.36 g, 44.2mmol) in anhydrous acetonitrile (1 L) was stirred at room temperaturefor 1 hour. Compound 20 (3.40 g, 14.6 mmol) was added to the suspension,and then DIEA (11.5 g, 89.0 mmol) was added dropwise. The mixture wasstirred at 45° C. for 4 days. The resulting precipitate was filtered andthoroughly washed with DCM. The filtrate was concentrated to dryness andthe residue was dissolved in DCM (200 mL) and washed with 1N HCl (200mL×2) followed by 5% aqueous NaHCO₃ (200 mL×3) and then brine (200mL×1). The mixture was then dried over Na₂SO₄ and concentrated in vacuo.The residue was purified via column chromatography on silica gel(CH₂Cl₂/MeOH 100:1-*50:1) to give compound 21 as a light yellow solid.¹H-NMR (400 MHZ, DMSO-d6) δ 12.96 (d J 6.4 Hz, 1H); 12.1 (s, 1H); 8.9(d, J 6.4 Hz, 1H); 8.33 (d, J 8 Hz, 1H); 7.84-7.75 (m, 2H); 7.55-7.48(m, 3H); 1.47 (s, 6H); 1.45 (s, 9H).

Procedure for the preparation of2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phenyl)-2-methylpropanoicacid (28)

To a stirred solution of compound 21 (0.9 g, 2.45 mmol) in MeOH (50 mL)was added NaOH (1.5 g, 37.5 mmol) at 0° C. After stirring for 16 hoursat 40° C., the solvent was evaporated in vacuo, then the residue wasdissolved in H₂O (50 ml). The precipitate was filtered and the filtratewas washed with DCM (100 mL×1) and ethyl acetate (100 mL×1). The aqueouslayer was acidified with 2N HCl to pH 1-2. The precipitate was filteredand washed with H₂O (80 mL) and heptane (50 mL). It was dried in vacuoto give compound 28 as a white solid. ¹H NMR (DMSO-d₆; 400 MHz) δ 12.85(s), δ 11.84 (s), δ 11.77 (s), δ 9.39 (s), δ 8.86 (s), δ 8.33 (s), δ7.79 (m), δ 7.52 (m), δ 7.18 (s), δ 7.09 (s), δ 1.44 (s), δ 1.40 (s). MSfound (M+H) 423.08.

Example 4: Second alternative Synthesis ofN-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(27)

A 3-neck 50 mL round bottom flask was equipped with magnetic stirrer,nitrogen bubbler and thermocouple. Compound 21 (514 mg, 1.27 mmol) and2-MeTHF (4 mL) are charged to the flask. The reaction mixture wasstirred at room temperature. Lithium aluminum hydride (204 mg, 6.6 mmol)was added as solid until 100% conversion is achieved, which wasmonitored using HPLC. Potassium sodium 2,3-dihydroxybutanedioatetetrahydrate salt (50 mL of a 400 g/L solution) and MTBE (50 mL) wereadded to the reaction mixture. The resulting solution was stirred for 15minutes and then let sit for 15 min. The organic layer was separated andthe pH of the aqueous layer was adjusted to a pH of about 6-7 by addingTartaric acid. The aqueous layer was extracted with MTBE. The organiclayer was concentrated and dried under high vacuum to provide the titlecompound as an off-white powder. The ¹H-NMR spectrum was consistent withthat reported above.

Example 5: Alternative Total synthesis of2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phenyl)-2-methylpropanoicacid (28)

Procedure for the preparation of Carbonic acid 2-bromide, 4-tertbutylphenyl ester methyl ester (35)

A 3-neck 2 L round bottom flask was equipped with mechanical stirrer,nitrogen bubbler and thermocouple. 2-Bromo-4-tertbutyl phenol (50 g,211.7 mmol) was added followed by DCM (1.75 L), DMAP (1.29 g, 10.58mmol) and Et₃N (44.3 mL, 317.6 mmol). The reaction mixture was cooleddown to 0° C. Methyl chloroformate (19.62 mL, 254 mmol) was addeddrop-wise to the reaction mixture. The mixture was allowed to warm toroom temperature while stirring overnight. When the reaction wascomplete, the mixture was filtered via sintered funnel. The filtrate wastransferred into 1 L separatory funnel. To quench, 1N HCl (300 mL) wasadded to filtrate and the organic layer was separated. The organic layerwas then washed with a mixture of 291 mL saturated NaHCO₃ and 100 mLwater. The layers were separated, and the aqueous layer was determinedto have a pH of about 8. The organic layer was concentrated and driedunder high vacuum for about 16 hours to give the title compound as aclear yellow oil, which was used in the next step without furtherpurification. ¹H-NMR (400 MHz. DMSO-d6) 7.66 (d, J 2.0 Hz, 1H), 7.46(dd, J 8.4, 2.0 Hz, 1H), 7.32 (d, J 8.4 Hz, 1H), 3.86 (s, 3H), 1.28 (s,9H)

Procedure for the preparation of (2-bromo-4-tert-butyl-5-nitro-phenyl)methyl carbonate (36)

A 3-neck 2 L round bottom flask was equipped with mechanical stirrer,nitrogen bubbler and thermocouple. Compound 35 (176 g, 612.9 mmol) andconcentrated sulfuric acid (264 mL) were charged to the flask. Thereaction mixture was cooled to −5° C.-0° C. Nitric acid (28.6 mL, 612.9mmol) was added drop-wise and the reaction mixture was stirred at 0° C.for 2 hours. When complete, water (264 mL) was added followed by MTBE(264 mL). The solution was stirred for 15 minutes, then let stand for 15minutes. The organic layer was separated, concentrated and dried underhigh vacuum to give the title compound as a dark brown oil, which wasused in the next step without further purification. ¹H-NMR (400 MHz.DMSO-d6) 7.96 (s, 1H), 7.92 (s, 1H), 3.89 (s, 3H), 1.34 (s, 9H)

Procedure for the preparation of 2-bromo-4-tert-butyl-5-nitro-phenol(37)

(2-Bromo-4-tert-butyl-5-nitro-phenyl)methyl carbonate (72.9 g, 219.5mmol) was charged to a reactor and DCM (291.6 mL) was added. The yellowreaction solution was cooled using an ice bath. Sodium methoxide (67.04g, 69.11 mL of 5.4 M, 373.2 mmol) was added portion-wise at 2.2-6.9 OC.After complete addition, the reaction was slowly warmed to ambienttemperature. When complete, the reaction was cooled to 0° C. andquenched with 1M HCl (373.2 mL, 373.2 mmol). The biphasic mixture wasstirred for 20 min and transferred to a seperatory funnel. The organiclayer was separated and washed with water (300 mL) followed by brine(300 ml). The organic layer was concentrated and the crude product driedunder high vacuum. The product was further purified using SupercriticalFluid Chromatography (SFC) separation on a Berger MultiGram III (MettlerToledo AutoChem, Newark Del.). The method conditions were 20% methanolat 250 mL/min on a PPU column (30*150) from Princeton Chromatography,100 bar, 35 C, 220 nm. An injection of 3.5 mL of a 55-70 mg/mL solutionwas injected. The data was collected using SFC ProNTo software. Thepurified product received from SFC purification was a methanol solvate.To remove the methanol, an azeotropic distillation was performed. Thedark yellow solid, 2-bromo, 4-tertbuyl, 5-nitro phenol methanol solvate,(111.3 g, 0.59.9 mmol) was charged to a 1 L round bottom flask, followedby heptane (500 mL). The slurry is heated to 64° C. to obtain a clearsolution. The solvent was distilled under reduced pressure (649 mbar)for 30 minutes and then stripped to dryness. This procedure was repeatedthree times until no MeOH was detected by ¹H-NMR. The product was driedunder high vacuum for 16 hours to give the product as a dark yellow semisolid. ¹H-NMR (400 MHZ, DMSO-d6) δ 11.2 (bs, OH), 7.69 (s, 1H); 7.03 (s,1H); 1.30 (s, 9H)

Procedure for the preparation of5-tert-butyl-3,3-dimethyl-6-nitrobenzofuran-2(3H)-one (19)

Difluorozinc (6.093 g, 58.92 mmol) was added to a round bottomed flask,which was flushed with nitrogen. Pd(tBu₃P)₂ (2 g, 3.835 mmol) was thenadded under nitrogen stream. 2-Bromo-4-tert-butyl-5-nitro-phenol (16.15g, 58.92 mmol) dissolved in DMF (80.75 mL) was then added to the flask.The reaction mixture was an orange suspension.(1-Methoxy-2-methyl-prop-1-enoxy)trimethylsilane (21.61 g, 25.13 mL,117.8 mmol) was added to the mixture and the resulting mixture washeated to 80° C. and stirred for 16 h. When complete, the reactionmixture was cooled to ambient temperature and filtered through Celite®.The filter cake was washed with MTBE (536.0 mL) and water (893.3 mL) wasadded to the filtrate. The mixture was stirred for 15 min and settledfor another 15 min. The layers were separated and 0.5M HCl (500 mL,250.0 mmol) was added to the organic phase. The layers were separatedand the organic layer was washed with water (500 mL). The layers wereseparated and the organic layer was washed with NaCl (500 mL; 8 wt %).The organic layer was separated and the solvent removed in vacuo. Thecrude product was obtained as a brown crystalline solid and was thenpurified through a silica plug, using hexane:MTBE 20:1-10:1 as aneluent. The fractions containing product were combined and the solventremoved in vacuo to give the pure product as a white crystalline solid.¹H-NMR (400 MHZ, DMSO-d6) δ 7.80 (s, 1H); 7.62 (s, 1H); 1.49 (s, 6H);1.34 (s, 9H)

Procedure for the preparation of6-amino-5-tert-butyl-3,3-dimethylbenzofuran-2(3H)-one (20)

Palladium on carbon (wet; 5 wt %) was placed into a round bottomed flaskunder nitrogen flow. 5-tert-butyl-3,3-dimethyl-6-nitro-benzofuran-2-one(4.7 g, 17.85 mmol) was then added to the vessel. Methanol (120 mL) wasthen carefully charged to the vessel under nitrogen atmosphere. Thevessel was then purged with N₂, evacuated, then charged with hydrogengas. The vessel was evacuated and re-charged with hydrogen gas, and thena continuous hydrogen gas stream was introduced. After completion, thereaction was filtered through Celite® and the cake was washed with MeOH(300 ml). The solvent was removed in vacuo and the product dried underhigh vacuum to give a white crystalline solid. ¹H-NMR (400 MHZ, DMSO-d6)a 7.05 (s, 1H); 6.48 (s, 1H); 5.02 (s, 2H, NH₂); 1.34 (s, 6H); 1.30 (s,9H)

Procedure for the preparation ofN-(5-tert-butyl-3,3-dimethyl-2-oxo-2,3-dihydrobenzofuran-6-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(21)

A reaction vessel was charged with compound 26 (2.926 g, 15.43 mmol),Compound 20 (4.32 g, 18.52 mmol), 2-MeTHF (35.99 mL), and subsequently50% T₃P in 2-MeTHF (13.36 g, 21.00 mmol). Pyridine (2.441 g, 2.496 mL,30.86 mmol) was added and the suspension heated at 47.5° C.±5° C. for 18h. After completion, the reaction was cooled to ambient temperature and2-MeTHF (36) and water (30 ml) were added. The layers were split and theorganic layer was washed with 10 wt % citric acid solution (30 ml),water (30 ml) and twice with NaHCO₃ (20 ml). The organic layer waswashed with brine (50 ml), separated and the solvent removed in vacuo.The crude product was dissolved in MTBE (100 ml) and hexane (200 ml) wasadded as an anti-solvent. A solid precipitated and the resulting slurrywas stirred for two hours. The solid was collected by suction filtrationand the cake was washed with hexane. The resulting product was dried ina vacuum oven at 55° C. with nitrogen bleed to give the title compoundas a beige solid. ¹H-NMR (400 MHZ, DMSO-d6) δ 12.96 (d J 6.4 Hz, 1H);12.1 (s, 1H); 8.9 (d, J 6.4 Hz, 1H); 8.33 (d, J 8 Hz, 1H); 7.84-7.75 (m,2H); 7.55-7.48 (m, 3H); 1.47 (s, 6H); 1.45 (s, 9H).

Procedure for the preparation of2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phenyl)-2-methylpropanoicacid (28)

Compound 26 (81.30 mg, 0.4288 mmol) and Compound 20 (110 mg, 0.4715mmol) were charged to a round bottomed flask. 2-MeTHF (1 mL) followed by50% T₃P in 2-MeTHF (371.4 mg, 0.5836 mmol) and pyridine (67.84 mg, 69.37μL, 0.8576 mmol) in 2-MeTHF were then added. The suspension was heatedat 47.5° C.±5° C. overnight. After completion, the reaction was cooledto ambient temperature. 2-MeTHF (1.014 mL) and water (811.2 μL) wereadded. The layers were separated and the organic layer was washed withwater (811.2 μL) and twice with NaHCO₃ (2 ml). The organic layer wastransferred into a round bottomed flask. LiOH (38.6 mg, 0.9 mmol)dissolved in water (2 mL) was added and the reaction was heated to 45°C. After completion, the layers were separated and the organic layer wasdiscarded. The aqueous layer was cooled with an ice bath andhydrochloric acid (10.72 mL of 1.0 M, 10.72 mmol) was added to thesolution until the pH reached a pH of about 3-4. The aqueous layer wasextracted twice with 2-MeTHF (5 ml), and the organic layers werecombined and washed with brine (5 ml). The organic layer was separatedand the solvent removed in vacuo. The resulting solid was dried in avacuum oven with nitrogen bleed at 50° C. to give the title compound.¹H-NMR (400 MHZ, DMSO-d6) δ 12.89 (d, J 6.8 Hz, 1H); 11.84 (s, 1H);11.74 (s, 1H); 9.36 (s, 1H); 8.87-8.61 (d, J 6.4 Hz, 1H); 8.34-8.32 (d,J 9.1 Hz 1H); 7.83-7.745 (m, 2H); 7.17-7.09 (m, 1H); 7.17 (s, 1H); 7.09(s, 1H); 1.43 (s, 6H); 1.40 (s, 9H).

Example 6: Total synthesis ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(34)

Procedure for the preparation of 2,4-di-tert-butylphenyl methylcarbonate (30)

Method 1

To a solution of 2,4-di-tert-butyl phenol, 29, (10 g, 48.5 mmol) indiethyl ether (100 mL) and triethylamine (10.1 mL, 72.8 mmol), was addedmethyl chloroformate (7.46 mL, 97 mmol) dropwise at 0° C. The mixturewas then allowed to warm to room temperature and stir for an additional2 hours. An additional 5 mL triethylamine and 3.7 mL methylchloroformate was then added and the reaction stirred overnight. Thereaction was then filtered, the filtrate was cooled to 0° C., and anadditional 5 mL triethylamine and 3.7 mL methyl chloroformate was thenadded and the reaction was allowed to warm to room temperature and thenstir for an addition 1 hours. At this stage, the reaction was almostcomplete and was worked up by filtering, then washing with water (2×),followed by brine. The solution was then concentrated to produce ayellow oil and purified using column chromatography to give compound 30.¹H NMR (400 MHz, DMSO-d₆) δ 7.35 (d, J=2.4 Hz, 1H), 7.29 (dd, J=8.4, 2.4Hz, 1H), 7.06 (d, J=8.4 Hz, 1H), 3.85 (s, 3H), 1.30 (s, 9H), 1.29 (s,9H).

Method 2

To a reactor vessel charged with 4-dimethylaminopyridine (DMAP, 3.16 g,25.7 mmol) and 2,4-ditert-butyl phenol (compound 29, 103.5 g, 501.6mmol) was added methylene chloride (415 g, 313 mL) and the solution wasagitated until all solids dissolved. Triethylamine (76 g, 751 mmol) wasthen added and the solution was cooled to 0-5° C. Methyl chloroformate(52 g, 550.3 mmol) was then added dropwise over 2.5-4 hours, whilekeeping the solution temperature between 0-5 OC. The reaction mixturewas then slowly heated to 23-28° C. and stirred for 20 hours. Thereaction was then cooled to 10-15° C. and charged with 150 mL water. Themixture was stirred at 15-20° C. for 35-45 minutes and the aqueous layerwas then separated and extracted with 150 mL methylene chloride. Theorganic layers were combined and neutralized with 2.5% HCl (aq) at atemperature of 5-20° C. to give a final pH of 5-6. The organic layer wasthen washed with water and concentrated in vacuo at a temperature below20° C. to 150 mL to give compound 30 in methylene chloride.

Procedure for the preparation of 5-nitro-2,4-di-tert-butylphenyl methylcarbonate (31)

Method 1

To a stirred solution of compound 30 (6.77 g, 25.6 mmol) was added 6 mLof a 1:1 mixture of sulfuric acid and nitric acid at 0° C. dropwise. Themixture was allowed to warm to room temperature and stirred for 1 hour.The product was purified using liquid chromatography (ISCO, 120 g, 0-7%EtOAc/Hexanes, 38 min) producing about an 8:1-10:1 mixture ofregioisomers of compound 31 as a white solid. ¹H NMR (400 MHz, DMSO-d₆)δ 7.63 (s, 1H), 7.56 (s, 1H), 3.87 (s, 3H), 1.36 (s, 9H), 1.32 (s, 9H).HPLC ret. time 3.92 min 10-99% CH₃CN, 5 min run; ESI-MS 310 m/z (MH)⁺.

Method 2

To compound 30 (100 g, 378 mmol) was added DCM (540 g, 408 mL). Themixture was stirred until all solids dissolved, and then cooled to −5-0°C. Concentrated sulfuric acid (163 g) was then added dropwise, whilemaintaining the initial temperature of the reaction, and the mixture wasstirred for 4.5 hours. Nitric acid (62 g) was then added dropwise over2-4 hours while maintaining the initial temperature of the reaction, andwas then stirred at this temperature for an additional 4.5 hours. Thereaction mixture was then slowly added to cold water, maintaining atemperature below 5° C. The quenched reaction was then heated to 25° C.and the aqueous layer was removed and extracted with methylene chloride.The combined organic layers were washed with water, dried using Na₂SO₄,and concentrated to 124-155 mL. Hexane (48 g) was added and theresulting mixture was again concentrated to 124-155 mL. More hexane (160g) was subsequently added to the mixture. The mixture was then stirredat 23-27° C. for 15.5 hours, and was then filtered. To the filter cakewas added hexane (115 g), the resulting mixture was heated to reflux andstirred for 2-2.5 hours. The mixture was then cooled to 3-7° C., stirredfor an additional 1-1.5 hours, and filtered to give compound 31 as apale yellow solid.

Procedure for the preparation of 5-amino-2,4-di-tert-butylphenyl methylcarbonate (32)

2,4-Di-tert-butyl-5-nitrophenyl methyl carbonate (1.00 eq) was chargedto a suitable hydrogenation reactor, followed by 5% Pd/C (2.50 wt % drybasis, Johnson-Matthey Type 37). MeOH (15.0 vol) was charged to thereactor, and the system was closed. The system was purged with N₂ (g),and was then pressurized to 2.0 Bar with H₂ (g). The reaction wasperformed at a reaction temperature of 25° C.+/−5° C. When complete, thereaction was filtered, and the reactor/cake was washed with MeOH (4.00vol). The resulting filtrate was distilled under vacuum at no more than50° C. to 8.00 vol. Water (2.00 vol) was added at 45° C.+/−5° C. Theresultant slurry was cooled to 0° C.+/−5. The slurry was held at 0°C.+/−5° C. for no less than 1 hour, and filtered. The cake was washedonce with 0° C.+/−5° C. MeOH/H₂O (8:2) (2.00 vol). The cake was driedunder vacuum (−0.90 bar and −0.86 bar) at 35° C.-40° C. to give compound32. ¹H NMR (400 MHz, DMSO-d₆) δ 7.05 (s, 1H), 6.39 (s, 1H), 4.80 (s,2H), 3.82 (s, 3H), 1.33 (s, 9H), 1.23 (s, 9H).

Once the reaction was complete, the resulting mixture was diluted withfrom about 5 to 10 volumes of MeOH (e.g., from about 6 to about 9volumes of MeOH, from about 7 to about 8.5 volumes of MeOH, from about7.5 to about 8 volumes of MeOH, or about 7.7 volumes of MeOH), heated toa temperature of about 35±5 OC, filtered, washed, and dried, asdescribed above.

Preparation ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(34)

4-Oxo-1,4-dihydroquinoline-3-carboxylic acid, 26, (1.0 eq) and5-amino-2,4-di-tert-butylphenyl methyl carbonate, 32, (1.1 eq) werecharged to a reactor. 2-MeTHF (4.0 vol, relative to the acid) was addedfollowed by T3P® 50% solution in 2-MeTHF (1.7 eq). The T3P chargedvessel was washed with 2-MeTHF (0.6 vol). Pyridine (2.0 eq) was thenadded, and the resulting suspension was heated to 47.5+/−5.0° C. andheld at this temperature for 8 hours. A sample was taken and checked forcompletion by HPLC. Once complete, the resulting mixture was cooled to25.0° C.+/−2.5° C. 2-MeTHF was added (12.5 vol) to dilute the mixture.The reaction mixture was washed with water (10.0 vol) 2 times. 2-MeTHFwas added to bring the total volume of reaction to 40.0 vol (−16.5 volcharged). To this solution was added NaOMe/MeOH (1.7 equiv) to performthe methanolysis. The reaction was stirred for no less than 1.0 hour,and checked for completion by HPLC. Once complete, the reaction wasquenched with 1 N HCl (10.0 vol), and washed with 0.1 N HCl (10.0 vol).The organic solution was polish filtered to remove any particulates andplaced in a second reactor. The filtered solution was concentrated at nomore than 35° C. (jacket temperature) and no less than 8.0° C. (internalreaction temperature) under reduced pressure to 20 vol. CH₃CN was addedto 40 vol and the solution concentrated at no more than 35° C. (jackettemperature) and no less than 8.0° C. (internal reaction temperature) to20 vol. The addition of CH₃CN and concentration cycle was repeated 2more times for a total of 3 additions of CH₃CN and 4 concentrations to20 vol. After the final concentration to 20 vol, 16.0 vol of CH₃CN wasadded followed by 4.0 vol of H₂O to make a final concentration of 40 volof 10% H₂O/CH₃CN relative to the starting acid. This slurry was heatedto 78.0° C.+/−5.0° C. (reflux). The slurry was then stirred for no lessthan 5 hours. The slurry was cooled to 0.0° C.+/−5° C. over 5 hours, andfiltered. The cake was washed with 0.0° C.+/−5.0° C. CH₃CN (5 vol) 4times. The resulting solid (compound 34) was dried in a vacuum oven at50.0° C.+/−5.0° C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.8 (s, 1H), 11.8 (s,1H), 9.2 (s, 1H), 8.9 (s, 1H), 8.3 (s, 1H), 7.2 (s, 1H), 7.9 (t, 1H),7.8 (d, 1H), 7.5 (t, 1H), 7.1 (s, 1H), 1.4 (s, 9H), 1.4 (s, 9H).

Alternative Preparation ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(34)

4-Oxo-1,4-dihydroquinoline-3-carboxylic acid, 26, (1.0 eq) and5-amino-2,4-di-tert-butylphenyl methyl carbonate, 32, (1.1 eq) werecharged to a reactor. 2-MeTHF (4.0 vol, relative to the acid) was addedfollowed by T3P® 50% solution in 2-MeTHF (1.7 eq). The T3P chargedvessel was washed with 2-MeTHF (0.6 vol). Pyridine (2.0 eq) was thenadded, and the resulting suspension was heated to 47.5+/−5.0° C. andheld at this temperature for 8 hours. A sample was taken and checked forcompletion by HPLC. Once complete, the resulting mixture was cooled to20° C.+/−5° C. 2-MeTHF was added (12.5 vol) to dilute the mixture. Thereaction mixture was washed with water (10.0 vol) 2 times and 2-MeTHF(16.5 vol) was charged to the reactor. This solution was charged with30% w/w NaOMe/MeOH (1.7 equiv) to perform the methanolysis. The reactionwas stirred at 25.0° C.+/−5.0° C. for no less than 1.0 hour, and checkedfor completion by HPLC. Once complete, the reaction was quenched with1.2 N HCl/H₂O (10.0 vol), and washed with 0.1 N HCl/H₂O (10.0 vol). Theorganic solution was polish filtered to remove any particulates andplaced in a second reactor.

The filtered solution was concentrated at no more than 35 OC (jackettemperature) and no less than 8.0° C. (internal reaction temperature)under reduced pressure to 20 vol. CH₃CN was added to 40 vol and thesolution concentrated at no more than 35° C. (jacket temperature) and noless than 8.0° C. (internal reaction temperature) to 20 vol. Theaddition of CH₃CN and concentration cycle was repeated 2 more times fora total of 3 additions of CH₃CN and 4 concentrations to 20 vol. Afterthe final concentration to 20 vol, 16.0 vol of CH₃CN was chargedfollowed by 4.0 vol of H₂O to make a final concentration of 40 vol of10% H₂O/CH₃CN relative to the starting acid. This slurry was heated to78.0° C.+/−5.0° C. (reflux). The slurry was then stirred for no lessthan 5 hours. The slurry was cooled to 20 to 25° C. over 5 hours, andfiltered. The cake was washed with CH₃CN (5 vol) heated to 20 to 25° C.4 times. The resulting solid (compound 34) was dried in a vacuum oven at50.0° C.+/−5.0° C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.8 (s, 1H), 11.8 (s,1H), 9.2 (s, 1H), 8.9 (s, 1H), 8.3 (s, 1H), 7.2 (s, 1H), 7.9 (t, 1H),7.8 (d, 1H), 7.5 (t, 1H), 7.1 (s, 1H), 1.4 (s, 9H), 1.4 (s, 9H).

Example 7: Procedure for the biosynthesis ofN-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(27) and2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phenyl)-2-methylpropanoicacid (28)

Streptomyces rimosus (DSM 40260) was purchased from DSMZ as frozenculture. This culture was used to inoculate agar slants, which weremaintained and stored at 4° C. Yeast extract-malt extract-peptone (YMP)media containing yeast extract (4 g/L), malt extract (10 g/L) and soyaflour (5 g/L) was prepared and sterilized at 130° C. for 60 minutes.Five flasks containing 1 L of YMP media were directly inoculated with S.rimosus from the agar slants. The culture was allowed to grow for 2-3days at 30° C. with gentle agitation of approximately 100 rpm. Underthese conditions, two growth types have been observed, either a cloudysolution or spherical particulates which aggregate at the bottom of theflask. The latter growth type has been shown to result in higherconversions to Compound 27. The cells were then spun down, harvested andresuspended in two flasks containing 1 L of 0.1 M potassium phosphatebuffer, pH 7.0. 5.0 g of Compound 34 in 50 mL N,N-dimethylformamide(DMF) were added to the flasks. The reactions proceeded for 24 hours at30° C. with gentle agitation of about 100 rpm at which point conversionsof 7.59% Compound 27 and 1.17% Compound 28 were indicated by HPLC.

Both flasks were combined, centrifuged at 3500 rpm for 10 minutes, andre-suspended in 500 mL of methanol. This suspension was stirredvigorously for 30 minutes and then spun down again at 6000 rpm for 10minutes. The organic layer was collected and the process was repeatedtwo times. The methanol extracts were concentrated in vacuo to yield2.50 g, 1.57 g and 1.11 g of solid material, respectively. The solidsfrom these extracts were shown to contain 74.78-91.96% Compound 34,7.66-19.73% Compound 27 and 0.39-5.49% Compound 28. In an effort to culloff a portion of Compound 34 from the bio-oxidation products, the solidsfrom the first two extractions were combined, suspended in 250 mLmethanol, agitated vigorously for 1 hour and vacuum filtered. WhileCompounds 27 and 28 were enriched in the filtrate (22.09 and 6.14%,respectively), the solids still also contained Compound 27 (8.96%) andCompound 28 (0.50%).

The methanol filtrate containing approximately 2.2 g of dissolved solidswas adsorbent onto 4.5 g of silica and purified by flash chromatographyusing a gradient of 100% dichloromethane to 88:12dichloromethane/methanol. Fractions containing Compound 27 wereconcentrated in vacuo and further dried via freeze-drying to obtain 130mg of Compound 27 (98.5% purity by HPLC). A fraction containing impureCompound 28 was also concentrated in vacuo to yield less than 10 mg ofsolid.

The cell pellet was re-suspended in 500 mL methanol and homogenized in aBeadBeater to break apart the cells and recover any remaining product.The organic layer was obtained by centrifuging the homogenizedsuspension at 6000 rpm for 10 minutes. This was added to the solidobtained from the third extraction and the filtered solids from theslurry enrichment of the first two extractions and slurried at refluxovernight. The slurry was then cooled and suction filtered to obtain1.99 g of solid. The solid was re-dissolved in 300 mL methanol which wasthen adsorbed onto approximately 5 g of silica and purified by flashchromatography using a gradient of 100% dichloromethane to 94:6dichloromethane/methanol to provide 820 mg of solid containing Compound34 and Compound 27 as well as other impurities. This was re-columnedusing a more gradual solvent gradient (100% DCM up to a mixture of 6%MeOH/94% DCM) to obtain an additional 89 mg of Compound 27. The ¹H-NMRspectrum was consistent with that reported above.

Example 8: Procedure for the recrystallization ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(34)

Compound 34 (1.0 eq) was charged to a reactor. 2-MeTHF (20.0 vol) wasadded followed by 0.1N HCl (5.0 vol). The biphasic solution was stirredand separated and the top organic phase was washed twice more with 0.1NHCl (5.0 vol). The organic solution was polish filtered to remove anyparticulates and placed in a second reactor. The filtered solution wasconcentrated at no more than 35° C. (jacket temperature) and no morethan 8.0° C. (internal reaction temperature) under reduced pressure to10 vol. Isopropyl acetate (IPAc) (10 vol) was added and the solutionconcentrated at no more than 35° C. (jacket temperature) and no morethan 8.0 OC (internal reaction temperature) to 10 vol. The addition ofIPAc and concentration was repeated 2 more times for a total of 3additions of IPAc and 4 concentrations to 10 vol. After the finalconcentration, 10 vol of IPAc was charged and the slurry was heated toreflux and maintained at this temperature for 5 hours. The slurry wascooled to 0.0° C.+/−5° C. over 5 hours and filtered. The cake was washedwith IPAc (5 vol) once. The resulting solid was dried in a vacuum ovenat 50.0° C.+/−5.0° C.

Example 9: General Procedure to Test Solubility at pH 7.4

A high throughput shake flask assay was used to determine solubility ofcompounds in pH 7.4 buffer. To calculate the concentration of compoundsin solution, two conditions per compound were run: 300 uM in 100% DMSOand 200 uM in pH 7.4 phosphate buffer with 2% DMSO present. Each samplewas left to shake overnight then injected onto HPLC-UV to determine peakarea using the following conditions: Phenomenex 00A-4251-B0—30×2.00 mmLuna 3u C18(2) 100 A column; 0.8 mL/min flow rate; 20 uL injectionvolume; HPLC grade water with 0.1% formic acid and HPLC gradeacetonitrile with 0.1% formic acid mobile phases; peak area determinedat 254 nm. Solubility in uM was calculated using the following equation:cone.=(peak area pH 7.4)/(peak area 300 uM DMSO standard condition)×300uM concentration of standard condition. Peaks of interest wereidentified in buffer conditions based on retention time (RT) of thelargest area peak in the 300 uM DMSO standard condition.

VI. Activity Assays Example 10: General Procedure for Activity Assays

Assays for Detecting and Measuring ΔF508-CFTR Potentiation Properties ofCompounds

Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The assay utilizes fluorescent voltage sensing dyes to measure changesin membrane potential using a fluorescent plate reader (e.g., FLIPR III,Molecular Devices, Inc.) as a readout for increase in functionalΔF508-CFTR in NIH 3T3 cells. The driving force for the response is thecreation of a chloride ion gradient in conjunction with channelactivation by a single liquid addition step after the cells havepreviously been treated with compounds and subsequently loaded with avoltage sensing dye.

Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. This HTS assay utilizes fluorescent voltagesensing dyes to measure changes in membrane potential on the FLIPR IIIas a measurement for increase in gating (conductance) of ΔF508 CFTR intemperature-corrected ΔF508 CFTR NIH 3T3 cells. The driving force forthe response is a Cl⁻ ion gradient in conjunction with channelactivation with forskolin in a single liquid addition step using afluorescent plate reader such as FLIPR III after the cells havepreviously been treated with potentiator compounds (or DMSO vehiclecontrol) and subsequently loaded with a redistribution dye.

Solutions

Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH.Chloride-free bath solution: Chloride salts in Bath Solution #1 aresubstituted with gluconate salts.

Cell Culture NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR areused for optical measurements of membrane potential. The cells aremaintained at 37° C. in 5% CO₂ and 90% humidity in Dulbecco's modifiedEagle's medium supplemented with 2 mM glutamine, 10% fetal bovine serum,1×NEAA, β-ME, 1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks.For all optical assays, the cells were seeded at ˜20,000/well in384-well matrigel-coated plates and cultured for 2 hours at 37° C.before culturing at 27° C. for 24 hours for the potentiator assay. Forthe correction assays, the cells are cultured at 27° C. or 37° C. withand without compounds for 16-24 hours. Electrophysiological Assays forassaying ΔF508-CFTR modulation properties of compounds.

1. Ussing Chamber Assay

Ussing chamber experiments were performed on polarized airway epithelialcells expressing ΔF508-CFTR to further characterize the ΔF508-CFTRmodulators identified in the optical assays. Non-CF and CF airwayepithelia were isolated from bronchial tissue, cultured as previouslydescribed (Galietta, L. J. V., Lantero, S., Gazzolo, A., Sacco, O.,Romano, L., Rossi, G. A., & Zegarra-Moran, O. (1998) In Vitro Cell. Dev.Biol. 34, 478-481), and plated onto Costar® Snapwell™ filters that werepre-coated with NIH3T3-conditioned media. After four days the apicalmedia was removed and the cells were grown at an air liquid interfacefor >14 days prior to use. This resulted in a monolayer of fullydifferentiated columnar cells that were ciliated, features that arecharacteristic of airway epithelia. Non-CF HBE were isolated fromnon-smokers that did not have any known lung disease. CF-HBE wereisolated from patients homozygous for ΔF508-CFTR.

HBE grown on Costar® Snapwell™ cell culture inserts were mounted in anUssing chamber (Physiologic Instruments, Inc., San Diego, Calif.), andthe transepithelial resistance and short-circuit current in the presenceof a basolateral to apical Cl⁻ gradient (I_(SC)) were measured using avoltage-clamp system (Department of Bioengineering, University of Iowa,IA). Briefly, HBE were examined under voltage-clamp recording conditions(V_(hold)=0 mV) at 37° C. The basolateral solution contained (in mM) 145NaCl, 0.83 K₂HPO₄, 3.3 KH₂PO₄, 1.2 MgCl₂, 1.2 CaCl₂, 10 Glucose, 10HEPES (pH adjusted to 7.35 with NaOH) and the apical solution contained(in mM) 145 NaGluconate, 1.2 MgCl₂, 1.2 CaCl₂, 10 glucose, 10 HEPES (pHadjusted to 7.35 with NaOH).

Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. Forskolin (10μM) and all test compounds were added to the apical side of the cellculture inserts. The efficacy of the putative ΔF508-CFTR potentiatorswas compared to that of the known potentiator, genistein.

2. Patch-Clamp Recordings

Total Cl⁻ current in ΔF508-NIH3T3 cells was monitored using theperforated-patch recording configuration as previously described (Rae,J., Cooper, K., Gates, P., & Watsky, M. (1991) J. Neurosci. Methods 37,15-26). Voltage-clamp recordings were performed at 22° C. using anAxopatch 200B patch-clamp amplifier (Axon Instruments Inc., Foster City,Calif.). The pipette solution contained (in mM) 150 N-methyl-D-glucamine(NMDG)-Cl, 2 MgCl₂, 2 CaCl₂, 10 EGTA, 10 HEPES, and 240 μg/mlamphotericin-B (pH adjusted to 7.35 with HCl). The extracellular mediumcontained (in mM) 150 NMDG-Cl, 2 MgCl₂, 2 CaCl₂, 10 HEPES (pH adjustedto 7.35 with HCl). Pulse generation, data acquisition, and analysis wereperformed using a PC equipped with a Digidata 1320 A/D interface inconjunction with Clampex 8 (Axon Instruments Inc.). To activateΔF508-CFTR, 10 μM forskolin and 20 μM genistein were added to the bathand the current-voltage relation was monitored every 30 sec.

Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in I_(ΔF508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1×pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

3. Single-Channel Recordings

Gating activity of wt-CFTR and temperature-corrected ΔF508-CFTRexpressed in NIH3T3 cells was observed using excised inside-out membranepatch recordings as previously described (Dalemans, W., Barbry, P.,Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G.,Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature 354, 526-528)using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.).The pipette contained (in mM): 150 NMDG, 150 aspartic acid, 5 CaCl₂, 2MgCl₂, and 10 HEPES (pH adjusted to 7.35 with Tris base). The bathcontained (in mM): 150 NMDG-Cl, 2 MgCl₂, 5 EGTA, 10 TES, and 14 Trisbase (pH adjusted to 7.35 with HCl). After excision, both wt- andΔF508-CFTR were activated by adding 1 mM Mg-ATP, 75 nM of the catalyticsubunit of cAMP-dependent protein kinase (PKA; Promega Corp. Madison,Wis.), and 10 mM NaF to inhibit protein phosphatases, which preventedcurrent rundown. The pipette potential was maintained at 80 mV. Channelactivity was analyzed from membrane patches containing ≦2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Compounds of Formula 1 are useful as modulators of ATP binding cassettetransporters.

Other Embodiments

All publications and patents referred to in this disclosure areincorporated herein by reference to the same extent as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference. Should themeaning of the terms in any of the patents or publications incorporatedby reference conflict with the meaning of the terms used in thisdisclosure, the meaning of the terms in this disclosure are intended tobe controlling. Furthermore, the foregoing discussion discloses anddescribes merely exemplary embodiments of the present invention. Oneskilled in the art will readily recognize from such discussion and fromthe accompanying drawings and claims, that various changes,modifications and variations can be made therein without departing fromthe spirit and scope of the invention as defined in the followingclaims.

What is claimed is:
 1. A compound having the formula:


2. A pharmaceutically acceptable salt of a compound having the formula: